Carbohydrate content of ctla4 molecules

ABSTRACT

The invention provides for mammalian cells capable of producing recombinant CTLA4-Ig and variants thereof. The invention also provides for compositions comprising CTLA4-Ig and formulations thereof The invention further provides for methods for mass-producing CTLA4-Ig from mammalian cells capable of producing this recombinant protein, and for purifying the CTLA4-Ig.

RELATED APPLICATIONS

The present patent application_is a divisional application of U.S.application Ser. No. 16/042,977, filed Jul. 23, 2018, which is adivisional of U.S. application Ser. No. 12/086,786 with 371(c) date ofJan. 27, 2009, which is the national phase application of InternationalApplication No. PCT/US2006/049074, filed Dec. 19,1 2006, which_claimsthe priority of U.S. Ser. No. 60/752,267, filed on Dec. 20, 2005, U.S.Ser. No. 60/849,543, filed on Oct. 5, 2006, and U.S. Ser. No.60/752,150, filed on Dec. 20, 2005, all of which are hereby incorporatedby reference in their entireties. This application also incorporates byreference in its entirety the patent application entitled “StableProtein Formulations” with Attorney Docket Number 10739 PCT filed onDec. 19, 2006.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3338.1350006_Sequence_listing_ST25.txt; Size: 83,658 bytes; and Date ofCreation: Jul. 23, 2018) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cytotoxic T lymphocyte antigen 4 (CTLA4), a member of the immunoglobulinsuperfamily, is a molecule expressed by activated T cells. CTLA4 issimilar to the T-cell co-stimulatory molecule CD28, and both moleculesbind to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs).However, CTLA4 transmits an inhibitory signal to T cells, whereas CD28transmits a stimulatory signal.

CTLA4-Ig molecules are fusion proteins of the ligand-binding domain ofcytotoxic T lymphocyte antigen 4 (CTLA4) and an immunoglobulin (Ig)heavy chain constant region. This soluble molecule exerts itsphysiological effects by binding to B7 antigens (CD80 and CD86) on thesurface of various antigen-presenting cells (APC), thus blocking thefunctional interaction of B7-1 and B7-2 with CD28 on the surface ofT-cells. This blockade results in the suppression of T-cell activation,and hence, the suppression of the immune response. CTLA4-Ig moleculescan therefore provide a method for inhibiting tissue and/or solid organtransplant rejections, as well as a therapeutic use for diseases ordisorders that relate to disregulated immune responses in general,including autoimmunity. For example, CTLA4-Ig molecules can suppress theproduction of anti-dsDNA antibodies and decrease nephritis in lupusprone mice; can reduce proteinuria and prolong survival in mice withadvanced nephriti; and can improve clinical outcomes for psoriasis andrheumatoid arthritis.

To improve the therapeutic usefulness of CTLA4-Ig molecules, it isimportant to determine molecular alterations that can be made to enhancethe efficacy of the molecule as an inhibitor of T cell stimulation, forexample, by increasing the avidity and potency of the molecule for B7antigens. An increase in the avidity and potency of CTLA4-Ig moleculesmay allow for administration of a decreased amount of CTLA4-Ig moleculesto a patient to achieve a desired therapeutic effect (i.e.,administration of a lower dose). An increase in the avidity and potencyof CTLA4-Ig molecules may also decrease the number of doses or thefrequency of doses that are administered to a patient to achieve adesired therapeutic effect.

SUMMARY OF THE INVENTION

The present invention relates to improved compositions and methods forproducing CTLA4-Ig compositions. The invention is directed to CTLA4-Igmolecules, improved compositions comprising CTLA4-Ig molecules, andimproved methods for producing (including mass-producing) CTLA4-Igmolecules and other recombinant proteins.

The invention includes any permutations and/or combinations of any ofthe elements and characteristics described herein, whether describedsingly or in certain combinations or permutations.

Cells: The invention provides for a clonal Chinese Hamster Ovary cellpopulation capable of producing CTLA4-Ig. The invention provides for aclonal Chinese Hamster Ovary cell population capable of producingCTLA4-Ig, each cell comprising 30 or more copies of a CTLA4-Igexpression cassette. The invention also provides for a clonal ChineseHamster Ovary cell population capable of producing CTLA4-Ig, each cellcomprising 30 or more copies of a CTLA4-Ig expression cassette, whereinthe 30 or more copies are integrated at a single site in the genome ofeach cell. The invention provides for a clonal Chinese Hamster Ovarycell population capable of producing CTLA4-Ig, wherein a CTLA4-Igexpression cassette is stable over about 105 passages. In oneembodiment, the CTLA4-Ig is encoded by an expression cassette comprisinga nucleic acid sequence described by Koduri R., et al. (Gene, 2001,280:87-95) and in U.S. Pat. Nos. 6,800,457 and 6,521,419, which arehereby incorporated by reference in their entireties. In anotherembodiment, the CTLA4-Ig is encoded by an expression cassette integratedinto a cell genome from the cell population at a specific locusdescribed by Koduri R., et al. (Gene, 2001, 280:87-95) and in U.S. Pat.Nos. 6,800,457 and 6,521,419, which are hereby incorporated by referencein their entireties. In one embodiment, the population comprises asub-population of cells comprising 33 or more copies of the CTLA4-Igexpression cassette, wherein the 33 or more copies are integrated at asingle site in the genome of each cell of the subpopulation.

The invention provides for a clonal Chinese Hamster Ovary cellpopulation capable of producing CTLA4-Ig, wherein at least 75% of thepopulation of cells has 30 or more copies of a CTLA4-Ig expressioncassette, wherein the 30 or more copies are integrated at a single sitein the genome of each cell of the 75% of the population. The inventionprovides for a clonal Chinese Hamster Ovary cell population capable ofproducing CTLA4-Ig, wherein at least 85% of the population of cells has30 or more copies of a CTLA4-Ig expression cassette, wherein the 30 ormore copies are integrated at a single site in the genome of each cellof the 85% of the population. The invention provides for a clonalChinese Hamster Ovary cell population capable of producing CTLA4-Ig,wherein at least 95% of the population of cells has 30 or more copies ofa CTLA4-Ig expression cassette, wherein the 30 or more copies areintegrated at a single site in the genome of each cell of the 95% of thepopulation. In one embodiment, the cell population is capable ofproducing greater than 0.5 or more grams of CTLA4-Ig protein per literof liquid culture, and wherein the CTLA4-Ig exhibits acceptablecarbohydrate characteristics, where the molar ratio of sialic acid toCTLA4-Ig is from about 6 to about 14 at a culture scale of 1,000 L ormore. In another embodiment, the cell population has been adapted toserum-free, chemically defined medium. In another embodiment, CTLA4-Igproduced from culture of the cell population has an extinctioncoefficient of 1.00±0.05 AU mL cm-1 mg-1. In another embodiment, thecell population, when grown in culture, is capable of producing CTLA4-Igpolypeptides, wherein: (a) about 90% of the CTLA4-Ig polypeptidescomprise an amino acid sequence of SEQ ID NO:2 beginning with themethionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 beginning with thealanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 ending with the lysineat residue number 383, (d) about 96% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 ending with the glycineat residue number 382; and optionally, (e) about less than 1% of theCTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2beginning with the methionine at residue number 25.

The invention provides for a progeny cell of the clonal cell, whereinthe progeny cell produces CTLA4-Ig. In one embodiment, the progeny cellis obtained from culturing the clonal parental cell over at least 5generations. In another embodiment, the progeny cell is obtained fromculturing a cell over at least 10 generations, over at least 20generations, over at least 40 generations, over at least 50 generations,over at least 75 generations, or over at least 100 generations. Theinvention provides for a cell line produced from the clonal cell. In oneembodiment, the cell line is clonal. The invention provides for a cellline capable of producing: (a) a CTLA4-Ig fusion protein having an aminoacid sequence of SEQ ID NO:10 (methionine at amino acid position 27 andglycine at amino acid position 382; FIGS. 1A and 1B); (b) a CTLA4-Igfusion protein having an amino acid sequence of SEQ ID NO: 7 (methionineat amino acid position 27 and lysine at amino acid position 383; FIGS.1A and 1B); (c) a CTLA4-Ig fusion protein having an amino acid sequenceof SEQ ID NO: 9 (alanine at amino acid position 26 and glycine at aminoacid position 382; FIGS. 1A and 1B); (d) a CTLA4-Ig fusion proteinhaving an amino acid sequence of SEQ ID NO: 6 (alanine at amino acidposition 26 and lysine at amino acid position 383; FIGS. 1A and 1B); (e)a CTLA4-Ig fusion protein having an amino acid sequence of SEQ ID NO:8(methionine at amino acid position 25 and glycine at amino acid position382; FIGS. 1A and 1B); or (f) a CTLA4-Ig fusion protein having an aminoacid sequence of SEQ ID NO:5 (methionine at amino acid position 25 andlysine at amino acid position 383; FIGS. 1A and 1B). In anotherembodiment, the cell line is capable of producing CTLA4-Ig fusionproteins, wherein: (a) about 90% of the CTLA4-Ig polypeptides comprisean amino acid sequence of SEQ ID NO:2 beginning with the methionine atresidue 27; (b) about 10% of the CTLA4-Ig polypeptides comprise theamino acid sequence of SEQ ID NO:2 beginning with the alanine at residuenumber 26; (c) about 4% of the CTLA4-Ig polypeptides comprise the aminoacid sequence of SEQ ID NO:2 ending with the lysine at residue number383, (d) about 96% of the CTLA4-Ig polypeptides comprise the amino acidsequence of SEQ ID NO:2 ending with the glycine at residue number 382;and optionally, (e) about less than 1% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 beginning with themethionine at residue number 25.

In one embodiment, the CTLA4-Ig fusion proteins, which are produced fromculturing the cell line, have an extinction coefficient of 1.00±0.05 AUmL cm-1 mg-1. The invention provides for a cell population derived fromthe clonal cell line. In an embodiment, the cell population consists ofat least one additional genetic change as compared to the originalclonal cell line and wherein the derived cell population is capable ofproducing CTLA4-Ig. In another embodiment, the cell population consistsof at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 10, at least 15, or at least 20 additionalgenetic changes as compared to the parental cell, and wherein thederived cell population is capable of producing CTLA4-Ig. In oneembodiment, the genetic change comprises at least one non-conservativemutation in the cellular genome or in the recombinant expressioncassette encoding CTLA4-Ig. In another embodiment, the genetic changecomprises at least one additional recombinant nucleic acid within thecell. In a further embodiment, the change comprises a mutation of thecellular genome. In another embodiment, the change comprises theaddition of a nucleic acid to either the cell genome or as a transnucleic acid, which encodes an anti-apoptotic polypeptide. In anotherembodiment, the anti-apoptotic polypeptide relates to glycosylation. Inanother embodiment, genetic change comprises at least one mutation ofthe cellular genome or of the recombinant expression cassette encodingCTLA4-Ig.

Compositions: The invention provides for a population of CTLA4-Igmolecules having an average molar ratio of sialic acid groups toCTLA4-Ig dimer or molecule of from about 6 to about 18. The inventionprovides for a population of CTLA4-Ig molecules having an average molarratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about8 to about 18. The invention provides for a population of CTLA4-Igmolecules having an average molar ratio of sialic acid groups toCTLA4-Ig dimer or molecule of from about 11 to about 18. The inventionprovides for a population of CTLA4-Ig molecules having an average molarratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about12 to about 18. The invention provides for a population of CTLA4-Igmolecules having an average molar ratio of sialic acid groups toCTLA4-Ig dimer or molecule of from about 13 to about 18. The inventionprovides for a population of CTLA4-Ig molecules having an average molarratio of sialic acid groups to CTLA4-Ig dimer or molecule of from about14 to about 18. The invention provides for a population of CTLA4-Igmolecules having an average molar ratio of sialic acid groups toCTLA4-Ig dimer or molecule of from about 15 to about 17. The inventionprovides for a population of CTLA4-Ig molecules having an average molarratio of sialic acid groups to CTLA4-Ig dimer or molecule of about 16.The invention provides for a population of CTLA4-Ig molecules, whereingreater than 95% of the molecules are CTLA4-Ig dimers. In oneembodiment, greater than 98% of the molecules are CTLA4-Ig dimers. Inanother embodiment, greater than 99% of the molecules are CTLA4-Igdimers. In another embodiment, greater than 99.5% of the molecules areCTLA4-Ig dimers. In another embodiment, from about 95% to about 99.5% ofthe molecules are CTLA4-Ig dimers and about 0.5% to about 5% of themolecules are CTLA4-Ig tetramers or high molecular weight species. Inanother embodiment, about 98.6% of the molecules are CTLA4-Ig dimers andabout 1.2% of the molecules are CTLA4-Ig tetramers or high molecularweight species and about less than 0.7% of the molecules are CTLA4-Igmonomers. The invention provides for a population consisting of CTLA4-Igdimers. The invention provides for a population of CTLA4-Ig molecules,wherein the population is substantially free of CTLA4-Ig monomer. Theinvention provides for a population of CTLA4-Ig molecules, wherein thepopulation is substantially free of CTLA4-Ig tetramer. The inventionprovides for a population of CTLA4-Ig monomer molecules substantiallyfree of CTLA4-Ig dimer and tetramer. In one embodiment, each monomer ofeach CTLA4-Ig dimer has at least 3 sialic acid groups. In anotherembodiment, each monomer of each CTLA4-Ig dimer has from at least 3sialic acid groups to at least 8 sialic acid groups. The inventionprovides for a purified population of CTLA4-Ig tetramer molecules, thepopulation being substantially free of CTLA4-Ig dimer, and optionallywherein the population comprises an amount that is greater than about100 grams. The invention provides for a purified population of CTLA4-Igtetramer molecules, the population being substantially free of CTLA4-Igmonomer, and optionally wherein the population comprises an amount thatis greater than about 100 grams. In one embodiment, each tetramermolecule comprises two pairs of CTLA4-Ig polypeptides, wherein eachpolypeptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 5-10, and wherein each member of the pair ofpolypeptides is covalently linked to the other member, and wherein thetwo pairs of polypeptides are non-covalently associated with oneanother. In another embodiment, each tetramer molecule is capable ofbinding to a CD80 or CD86. In a further embodiment, each tetramermolecule has at least a 2-fold greater avidity for CD80 or CD86 ascompared to a CTLA4-Ig dimer molecule. In another embodiment, eachtetramer molecule has at least a 2-fold greater inhibition of T cellproliferation or activation as compared to a CTLA4-Ig dimer molecule.The invention provides for a composition comprising CTLA4-Ig molecules,wherein the composition comprises dominant isoforms visualizable on anisoelectric focusing gel of CTLA4-Ig which have an isoelectric point,pI, less than or equal to 5.1 as determined by isoelectric focusing. Inone embodiment, the invention provides for a composition comprisingCTLA4-Ig molecules, wherein the composition comprises dominant isoformsvisualizable on an isoelectric focusing gel of CTLA4-Ig which have anisoelectric point, pI, less than or equal to 5.8 as determined byisoelectric focusing. In one embodiment, the pI increases afterneuraminidase treatment. In one embodiment, the composition comprisesdominant isoforms visualizable on an isoelectric focusing gel ofCTLA4-Ig which have an isoelectric point, pI, less than or equal to 5.7,5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, or 4.5 asdetermined by isoelectric focusing. In another embodiment, at least 40%of the CTLA4-Ig molecules exhibit an isoelectric point less than orequal to about 5.1 as determined by isoelectric focusing. In anotherembodiment, at least 70% of the CTLA4-Ig molecules exhibit anisoelectric point less than or equal to about 5.1 as determined byisoelectric focusing. In another embodiment, at least 90% of theCTLA4-Ig molecules exhibit an isoelectric point less than or equal toabout 2.5 as determined by isoelectric focusing. The invention providesfor a population of CTLA4-Ig molecules having a pI of from about 2.0±0.2to about 5.0±0.2. The invention provides for a population of CTLA4-Igmolecules having a pI of from about 4.0±0.2 to about 5.0±0.2. Theinvention provides for a population of CTLA4-Ig molecules having a pIfrom about 4.3±0.2 to about 5.0±0.2. The invention provides for apopulation of CTLA4-Ig molecules having a pI of about 3.3±0.2 to about4.7±0.2. The invention provides for a method for preparing acomposition, the composition comprising a CTLA4-Ig molecule with a pI offrom about 2.0±0.2 to about 5.0±0.2, the method comprising: (a)subjecting a mixture of CTLA4-Ig molecules to isoelectric focusing gelelectrophoresis, wherein a single band on the gel represents apopulation of CTLA4-Ig molecules with a particular pI, and (b) isolatingthe population of CTLA4-Ig molecules having a pI of from about 2.0±0.2to about 5.0±0.2 so as to prepare the composition.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of GlcNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule offrom about 15 to about 35. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of GalNAc per mole of CTLA4-Igdimer or to CTLA4-Ig molecule of from about 1.7 to about 3.6. Theinvention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of galcatose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule offrom about 8 to about 17. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of fucose per mole of CTLA4-Igdimer or to CTLA4-Ig molecule of from about 3.5 to about 8.3. Theinvention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of mannose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule offrom about 7.2 to about 22. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of sialic acid per mole ofCTLA4-Ig dimer or to CTLA4-Ig molecule of from about 6 to about 12.

The invention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of GlcNAc per mole ofCTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; and (b)an average molar ratio of sialic acid per mole of CTLA4-Ig dimer orCTLA4-Ig molecule from about 6 to about 12. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Igmolecule from about 15 to about 35; (b) an average molar ratio of GalNAcper mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 to about3.6; and (c) an average molar ratio of sialic acid per mole of CTLA4-Igdimer or CTLA4-Ig molecule from about 6 to about 12. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer orCTLA4-Ig molecule from about 15 to about 35; (b) an average molar ratioof GalNAc per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 toabout 3.6; (c) an average molar ratio of galcatose per mole CTLA4-Igdimer or CTLA4-Ig molecule from about 8 to about 17; and (d) an averagemolar ratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Igmolecule from about 6 to about 12. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Igmolecule from about 15 to about 35; (b) an average molar ratio of GalNAcper mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 1.7 to about3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimer orCTLA4-Ig molecule from about 8 to about 17; (d) an average molar ratioof fucose per mole CTLA4-Ig dimer or CTLA4-Ig molecule from about 3.5 toabout 8.3; and (e) an average molar ratio of sialic acid per mole ofCTLA4-Ig dimer or CTLA4-Ig molecule from about 6 to about 12. Theinvention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of GlcNAc per mole ofCTLA4-Ig dimer or CTLA4-Ig molecule from about 15 to about 35; (b) anaverage molar ratio of GalNAc per mole CTLA4-Ig dimer or molecule fromabout 1.7 to about 3.6; (c) an average molar ratio of galcatose per moleCTLA4-Ig dimer or molecule from about 8 to about 17; (d) an averagemolar ratio of fucose per mole CTLA4-Ig dimer or molecule from about 3.5to about 8.3; (e) an average molar ratio of mannose per mole CTLA4-Igdimer or molecule from about 7.2 to about 22; and (f) an average molarratio of sialic acid per mole of CTLA4-Ig dimer or CTLA4-Ig moleculefrom about 6 to about 12. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein composition exhibits an NGNAchromatogram peak of about 9.589+/−0.3 and an NANA chromatogram peak ofabout 10.543+/−0.3. The invention provides for a composition comprisingCTLA4-Ig molecules, wherein the CTLA-Ig molecules exhibit a carbohydrateprofile as shown in FIG. 67. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules exhibit acarbohydrate profile of Domains I-V (e.g., I-IV), wherein Domain Icomprises peaks which represent a-sialylated oligosaccharides, Domain IIcomprises peaks which represent mono-sialylated oligosaccharides, DomainIII comprises peaks which represent di-sialylated oligosaccharides, andDomain IV comprises peaks which represent tri-sialylatedoligosaccharides. Domain V comprises peaks that representtetra-sialyated oligosaccharides. In one embodiment, the difference inretention times of N-linked oligosaccharides between a first peak inDomain I and a main peak in Domain II is from about 22 to about 28minutes. The invention provides for a composition comprising CTLA4-Igdimer molecules, wherein at least 0.5% of the CTLA4-Ig dimer moleculesare cysteinylated. In one embodiment, at least 1.0% of the CTLA4-Igdimer molecules are cysteinylated. The invention provides for apopulation of CTLA4-Ig molecules, wherein the population exhibits a massspectrometry profile as shown in FIGS. 8A and 8B. The invention providesfor a population of CTLA4-Ig molecules, wherein the population exhibitsa capillary electrophoresis profile as shown in FIGS. 19 and 20. Theinvention provides for a composition of CTLA4-Ig molecules having anaverage molar ratio of sialic acid groups to CTLA4-Ig dimer of fromabout 6 to about 18. The invention provides for a CTLA4-Ig compositionobtained by any of the methods described herein. The invention providesfor a population of CTLA4-Ig molecules, wherein the molecules areglycosylated at an aparagine amino acid residue at position 102 of SEQID NO:2, an aparagine amino acid residue at position 134 of SEQ ID NO:2,an aparagine amino acid residue at position 233 of SEQ ID NO:2, a serineamino acid residue at position 155 of SEQ ID NO:2, or a serine aminoacid residue at position 165 of SEQ ID NO:2.

The invention provides for a population of CTLA4-Ig molecules, whereinthe population of molecules is characterized by: (a) an average molarratio of GlcNAc per mole of CTLA4-Ig dimer or CTLA4-Ig molecule fromabout 15 to about 35; (b) an average molar ratio of GalNAc per moleCTLA4-Ig dimer or molecule from about 1.7 to about 3.6; (c) an averagemolar ratio of galcatose per mole CTLA4-Ig dimer or molecule from about8 to about 17; (d) an average molar ratio of fucose per mole CTLA4-Igdimer or molecule from about 3.5 to about 8.3; (e) an average molarratio of mannose per mole CTLA4-Ig dimer or molecule from about 7.2 toabout 22; (f) an average molar ratio of sialic acid per mole of CTLA4-Igdimer or molecule from about 6 to about 12; (g) a pI as determined fromvisualization on an isoelectric focusing gel in a range from about2.4±0.2 to about 5.0±0.2; (h) MCP-1 of less than or equal to 5 ppm; (i)less than 3.0% tetramer (e.g., 2.5% high molecular weight species ortetramer, 2.0% high molecular weigh species or tetramer; (j) less than0.5% monomer; (k) CTLA4-Ig polypeptides of the population having anamino acid at least 95% identical to any of SEQ ID NOS: 2-8; (1) whereinCTLA4-Ig molecules within the population is capable of binding to CD80and CD86.

Compositions: The invention provides for a composition comprising aneffective amount of the CTLA4-Ig molecules of the invention and apharmaceutically acceptable carrier. The invention provides for acomposition comprising excipients as described in U.S. Application No.60/752,150, filed Dec. 20, 2005. In one embodiment, the compositionincludes CTLA4-Ig. In one embodiment, the composition further comprisesa pharmaceutically acceptable diluent, adjuvant or carrier. In anotherembodiment, the composition further comprises maltose, sodium phosphatemonobasic monohydrate, sodium chloride, sodium hydroxide, and sterilewater. In another embodiment, the composition comprises sucrose,poloxamer, sodium phosphate monobasic monohydrate, sodium phosphatedibasic anhydrous, sodium chloride, sodium hydroxide, and sterile water.

Formulations and Kits: The invention provides for a lyophilized CTLA4-Igmixture comprising at least 95% CTLA4-Ig dimer, and not more than 5%CTLA4-Ig tetramer. In one embodiment, the mixture comprises at least 98%CTLA4-Ig dimer and no more than 2% CTLA4-Ig high molecular weightspecies or tetramer. In another embodiment, the mixture comprises atleast 99% CTLA4-Ig dimer and no more than 1% CTLA4-Ig high molecularweight species or tetramer. In another embodiment, the mixture comprisesat least 8.0 moles of sialic acid per mole of CTLA4-Ig dimer ormolecule. In another embodiment, the mixture comprises from about 15.7to about 31 moles of GlcNAc per mole of CTLA4-Ig dimer or molecule. Inanother embodiment, the mixture comprises from about 1.6 to about 3.2moles of GalNAc per mole of CTLA4-Ig dimer or molecule. In anotherembodiment, the mixture comprises from about 9.3 to about 15.5 moles ofgalactose per mole of CTLA4-Ig dimer or molecule. In one embodiment, themixture comprises from about 3.6 to about 7.9 moles of fucose per moleof CTLA4-Ig dimer or molecule. In one embodiment, the mixture comprisesfrom about 9.7 moles of mannose per mole of CTLA4-Ig dimer or molecule.The invention also provides for a pharmaceutical kit comprising: (a) acontainer containing a lyophilized CTLA4-Ig mixture of the invention;and (b) instructions for reconstituting the lyophilized CTLA4-Ig mixtureinto solution for injection.

Illustrative Methods of Treatment: The invention provides for a methodfor inhibiting T cell proliferation (or activation), the methodcomprising contacting a T cell with an effective amount of a CTLA4-Igcomposition of the invention. The invention provides for a method forinhibiting an immune response in a subject, the method comprisingadministering to a subject in need thereof an effective amount of aCTLA4-Ig composition of the invention. The invention provides for amethod for inducing immune tolerance to an antigen in a subject, themethod comprising administering to a subject in need thereof aneffective amount of a CTLA4-Ig composition of the invention. Theinvention provides for a method for treating inflammation in a subject,the method comprising administering to a subject in need thereof aneffective amount of a CTLA4-Ig composition of the invention. Theinvention provides for a method for treating rheumatoid arthritiscomprising administering to a subject in need thereof an effectiveamount of a CTLA4-Ig composition of the invention. The inventionprovides for a method for treating psoriasis in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a CTLA4-Ig composition of the invention. The inventionprovides for a method for treating lupus in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a CTLA4-Ig composition of the invention. The inventionprovides for a method for treating or preventing an allergy in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides for a method for treating or preventing graft vshost disease in a subject, the method comprising administering to asubject in need thereof an effective amount of a CTLA4-Ig composition ofthe invention. The invention provides for a method for treating orpreventing rejection of a transplanted organ in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a CTLA4-Ig composition of the invention. The inventionprovides for a method for treating multiple sclerosis in a subject, themethod comprising administering to a subject in need thereof aneffective amount of a CTLA4-Ig composition of the invention. Theinvention provides for a method for treating Crohn's Disease in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides a method for treating type I diabetes in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides a method for treating inflammatory bowel diseasein a subject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides a method for treating oophoritis in a subject,the method comprising administering to a subject in need thereof aneffective amount of a CTLA4-Ig composition of the invention. Theinvention provides a method for treating glomerulonephritis in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides a method for treating allergic encephalomyelitisin a subject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.The invention provides a method for treating myasthenia gravis in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the invention.

The invention provides for the use of a population of CTLA4-Ig moleculeshaving an average molar ratio of sialic acid groups to CTLA4-Ig dimer ormolecule of from about 6 to about 18 in the manufacture of a medicamentfor the therapeutic and/or prophylactic treatment of an immune disorder.The invention provides for the use of a population of CTLA4-Ig moleculeshaving an average molar ratio of sialic acid groups to CTLA4-Ig dimer ormolecule of from about 6 to about 18 in the manufacture of ananti-rheumatoid arthritis agent in a package together with instructionsfor its use in the treatment of rheumatoid arthritis.

Illustrative Combination therapies: The invention provides for a methodfor inhibiting T cell proliferation (or activation), the methodcomprising contacting a T cell with an effective amount of a CTLA4-Igcomposition of the invention in combination with methotrexate. Theinvention provides a method for inhibiting an immune response in asubject, the method comprising administering to a subject in needthereof an effective amount of a CTLA4-Ig composition of the inventionin combination with methotrexate. The invention provides a method forinducing immune tolerance to an antigen in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a CTLA4-Ig composition of the invention in combination withmethotrexate.

Methods for Producing CTLA4-Ig: The invention provides a method forproducing a recombinant protein, the method comprising: (a) expandingmammalian cells that secrete a recombinant protein, wherein theexpanding is from a seed culture to a liquid culture, wherein therecombinant protein concentration is at least 0.5 grams/L of liquidculture; and (b) isolating the recombinant protein from the liquidculture. The liquid culture can be at least 1,000 L, at least 5,000 L,at least 10,000 L, at least 15,000 L, at least 20,000 L, at least 25,000L, at least 30,000 L, at least 40,000 L. In one embodiment, theexpanding of step (a) comprises: (i) culturing the cells in aserum-free, chemically defined medium with at least four passages so asto obtain a cell density of at least about 1.0×10⁵ viable cells per mL,wherein each seed stage starts at about 2×10⁵ per ml and goes to 1-2 milcells per ml; (ii) maintaining the cells in culture for a timesufficient to produce from the culture at least about 0.5 g/L. In oneembodiment, the protein is a glycoprotein. In one embodiment, theprotein is a CTLA4-Ig protein. In one embodiment, the mammalian cellsare progeny of a CHO clonal cell line capable of producing CTLA4-Igfusion protein, wherein the CHO cells have stably integrated in theirgenome at least 30 copies of a CTLA4-Ig expression cassette. In oneembodiment, the time sufficient is a time by which the cells' viabilitydoes not fall below 30%. In another embodiment, the time sufficient is atime by which the cells' viability does not fall below 40%. In anotherembodiment, the time sufficient is a time by which the cells' viabilitydoes not fall below 50%. In another embodiment, the time sufficient is atime by which the cells' viability does not fall below 60%. In anotherembodiment, the time sufficient is a time by which the cells' viabilitydoes not fall below 70%, or 80% or 90% or 95%.

In a further embodiment, the at least four passages comprises: (i)growing the cells in a culture volume of at least 50 mL until a celldensity of from about 1 million to about 2.5 mill cells per ml isreached, (ii) growing the cells in a culture volume of at least 10 Luntil a cell density of about 1 million to about 2.5 million cells perml is reached; (iii) growing the cells in a culture volume of at least100 L until a cell density of about 1 million to about 2.5 million cellsper ml is reached; and (iv) growing the cells in a culture volume of 200L until a cell density of about 1 million to about 2.5 million cells perml is reached. In one embodiment, galactose is added to the serum-free,chemically defined medium. In one embodiment, the maintaining comprises(i) lowering the temperature of the culture from 37±2° C. to 34±2° C.;and (ii) lowering the temperature of the culture from 34±2° C. to 32±2°C. In another embodiment, the temperature is kept within the range of32±2° C. for at least 5 days. In another embodiment, the temperature iskept within the range of 32±2° C. for at least 6 days. In anotherembodiment, the temperature is kept within the range of 32±2° C. for atleast 7 days. In another embodiment, the temperature is kept within therange of 32±2° C. for at least 8 days. In another embodiment, thetemperature is kept within the range of 32±2° C. for at least 9 days. Inanother embodiment, the temperature is kept within the range of 32±2° C.for at least 10 days. In another embodiment, the temperature is keptwithin the range of 32±2° C. for at least 11 days. In anotherembodiment, the temperature is kept within the range of 32±2° C. for atleast 12 days. In another embodiment, the temperature is kept within therange of 32±2° C. for at least 13 days. In another embodiment, thetemperature is kept within the range of 32±2° C. for at least 14 days.In another embodiment, the temperature is kept within the range of 32±2°C. for at least 15 days. In another embodiment, the temperature is keptwithin the range of 32±2° C. for at least 16 days. In anotherembodiment, the temperature is kept within the range of 32±2° C. for atleast 17 days. In another embodiment, the temperature is kept within therange of 32±2° C. for at least 18 days. In another embodiment, thetemperature is kept within the range of 32±2° C. for up to 18 days. Inanother embodiment, the temperature is kept within the range of 32±2° C.until the cell density of the culture is from about 30×10⁵ to about79×10⁵ cells per mL of liquid culture.

The invention provides for a method for producing a recombinant protein,the method comprising: (a) expanding mammalian cells that secrete arecombinant protein from a seed culture to a liquid culture so that therecombinant protein concentration is at least 0.5 grams/L of liquidculture; and (b) isolating the recombinant protein from the liquidculture, wherein the isolating occurs only when the liquid culturecontains greater than or equal to about 6.0 moles of NANA per mole ofCTLA4-Ig protein or dimer. The invention provides for a method forproducing a recombinant protein, the method comprising: (a) expandingmammalian cells that secrete a recombinant protein from a seed cultureto a liquid culture of so that the recombinant protein concentration isat least 0.5 grams/L of liquid culture; and (b) isolating therecombinant protein from the liquid culture, wherein the isolatingoccurs only when the liquid culture has a cell density of from about33×10⁵ to about 79×10⁵ cells per mL. The invention provides for a methodfor producing a recombinant protein, the method comprising: (a)expanding mammalian cells that secrete a recombinant protein from a seedculture to a liquid culture so that the recombinant proteinconcentration is at least 0.5 grams/L of liquid culture; and (b)isolating the recombinant protein from the liquid culture, wherein theisolating occurs when cell viability in the liquid culture has notfallen below about 20%, or about 30%, or about 38%. The inventionprovides for a method for producing a recombinant protein, the methodcomprising: (a) expanding mammalian cells that secrete a recombinantprotein from a seed culture to a liquid culture of at least 10,000 L sothat the recombinant protein concentration is at least 0.5 grams/L ofliquid culture; and (b) isolating the recombinant protein from theliquid culture, wherein the isolating occurs only when endotoxin is lessthan or equal to about 76.8 EU per mL of liquid culture. The inventionprovides for a method for producing a recombinant protein, the methodcomprising: (a) expanding mammalian cells that secrete a recombinantprotein from a seed culture to a liquid culture of at least 10,000 L sothat the recombinant protein concentration is at least 0.5 grams/L ofliquid culture; and (b) isolating the recombinant protein from the atleast 10,000 L liquid culture, wherein the isolating occurs only whenbioburden is less than 1 colony forming unit per mL of liquid culture.The liquid culture of the invention can be of a volume of at least 5,000L, at least 10,000 L, at least 15,000 L, at least 20,000 L, at least25,000 L, at least 30,000 L, at least 40,000 L, at least 50,000 L, atleast 60,000 L.

The invention provides a method for producing a recombinant protein, themethod comprising: (a) expanding mammalian cells that secrete arecombinant protein from a seed culture to a liquid culture so that therecombinant protein concentration is at least 0.5 grams/L of liquidculture; and (b) isolating the recombinant protein from the liquidculture, wherein the isolating occurs only if at least two of thefollowing conditions are met: (i) the liquid culture contains greaterthan or equal to about 6.0 moles of NANA per mole of protein, (ii) theliquid culture has a cell density of from about 33×10⁵ to about 79×10⁵cells per mL,(iii) cell viability in the liquid culture has not fallenbelow about 20%, or about 38%, or (iv) amount of CTLA4-Ig in the cultureis greater than 0.5 g/L. In one embodiment, the isolating comprises: (i)obtaining a cell culture supernatent; (ii) subjecting the supernatant toanion exchange chromotagraphy to obtain an eluted protein product; (iii)subjecting the eluted protein product of step (ii) to hydrophobicinteraction chromatography so as to obtain an enriched protein product;(iv) subjecting the enriched protein product to affinity chromatographyto obtain an eluted and enriched protein product; and (v) subjecting theeluted and enriched protein product of (iv) to anion exchangechromatography. In another embodiment, the enriched protein productobtained in step (iii) is characterized in that a percentage of any highmolecular weight contaminant is less than 25% by weight. In anotherembodiment, the anion exchange chromatography of step (ii) is carriedout using a wash buffer comprising about 75 mM HEPES, and about 360 mMNaCl, and having a pH of about 8.0. In another embodiment, the anionexchange chromatography of step (ii) is carried out using an elutionbuffer comprising about 25 mM HEPES, and about 325 mM NaCl, and having apH of about 7.0. In another embodiment, the hydrophobic interactionchromatography of step (iii) is carried out using a single wash buffercomprising about 25 mM HEPES, and about 850 mM NaCl, and having a pH ofabout 7.0. In another embodiment, the affinity chromatography of step(iv) is carried out using a wash buffer comprising about 25 mM Tris, andabout 250 mM NaCl, and having a pH of about 8.0. In another embodiment,the affinity chromatography of step (iv) is carried out using an elutionbuffer comprising about 100 mM Glycine and having a pH of about 3.5. Inanother embodiment, the anion exchange chromatography of step (v) iscarried out using a wash buffer comprising about 25 mM HEPES, and fromabout 120 mM NaCl to about 130 mM NaCl, and having a pH of about 8.0. Inanother embodiment, the anion exchange chromatography of step (v) iscarried out using an elution buffer comprising about 25 mM HEPES, andabout 200 mM NaCl, and having a pH of about 8.0. In another embodiment,the anion exchange chromatography of step (ii) is carried out using acolumn having an anion exchange resin having a primary, secondary,tertiary, or quartenary amine functional group. In another embodiment,the resin has a quartenary amine functional group. In anotherembodiment, the hydrophobic interaction chromatography of step (iii) iscarried out using a hydrophobic interaction resin having a phenyl, anoctyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group. Inanother embodiment, the functional group is a phenyl functional group.In another embodiment, the affinity chromatography of step (iv) iscarried out using a column containing Protein A.

The invention provides for a method for preparing CTLA4-Ig, the methodcomprising purifying CTLA4-Ig from a liquid cell culture so that thepurified CTLA4-Ig (a) has about 38 ng of MCP-1 per mg of CTLA4-Ig dimer,and (b) comprises less than 2.5% of CTLA4-Ig high molecular weightspecies (e.g., tetramer) by weight. The invention provides for a methodfor producing CTLA4-Ig, the method comprising: (a) expanding progenycells or CHO cells that are capable of producing CTLA4-Ig, wherein theexpanding is from a seed culture to a liquid culture of at least 10,000L, wherein the CTLA4-Ig concentration is at least 0.5 grams/L of liquidculture; and (b) isolating CTLA4-Ig from the at least 10,000 L liquidculture, wherein the chromotagraphy is on a column with hydrophobicinteraction resin with at least a phenyl functional group, wherein theisolating comprises a step of hydrophobic interaction chromatographycarried out using a single wash buffer comprising about 25 mM HEPES, andabout 850 mM NaCl, and having a pH of about 7.0.

CTLA4-Ig molecules include beta polypeptide molecules.CTLA4^(A29YL104E)-Ig is a beta polypeptide molecule. The presentinvention relates to methods for producing (including mass-producing)beta polypeptide compositions or beta polypeptide molecule compositions,and improved compositions. The invention is directed to beta polypeptidemolecules, improved compositions comprising beta polypeptide molecules,and improved methods for producing (including mass-producing) betapolypeptide molecules and other recombinant glycoproteins.

Methods for producing beta polypeptides and other glycoproteins: Theinvention provides for a method for producing a recombinantglycoprotein, the method comprising: (a) expanding mammalian cells thatsecrete a recombinant glycoprotein, wherein the expanding is from a seedculture to a liquid culture of at least about 10,000 L, wherein therecombinant protein concentration is at least about 0.5 g/L of liquidculture, wherein the expanding comprises: (i) culturing the cells in aserum-free, chemically defined medium with at least four passages so asto obtain a cell density of at least about 1.0×10⁵ viable cells per mL,wherein each seed stage starts at about 2×10⁵ per ml and goes to about1-2 million cells per ml, wherein the culturing comprises: (1) culturingthe cells in a serum-free, chemically-defined inoculum medium for fromabout 15 days to about 25 days; then (2) culturing the cells in aserum-free, chemically-defined basal medium until a cell density ofabout at least 4 million cells per mL is reached; and (ii) maintainingthe cells in culture for a time sufficient to produce the recombinantprotein from the culture at least about 0.5 g/L; and (b) isolating therecombinant protein from the at least about 10,000 L liquid culture.

The invention provides for a method for producing a recombinantglycoprotein, the method comprising: (a) expanding mammalian cells thatsecrete a recombinant glycoprotein, wherein the expanding is from a seedculture to a liquid culture of at least about 10,000 L, wherein therecombinant protein concentration is at least about 0.5 g/L of liquidculture, wherein the expanding comprises: (i) culturing the cells in aserum-free, chemically defined medium with at least four passages so asto obtain a cell density of at least about 1.0×10⁵ viable cells per mL,wherein each seed stage starts at about 2×10⁵ per ml and goes to about1-2 million cells per ml; and (ii) maintaining the cells in culture fora time sufficient to produce the recombinant protein from the culture atleast about 0.5 g/L, wherein the maintaining comprises: (1) lowering thetemperature of the culture from 37±2° C. to 34±2° C.; and (2) adding apolyanionic compound to the culture; and (b) isolating the recombinantprotein from the at least about 10,000 L liquid culture.

The invention provides for a method for producing a recombinantglycoprotein, the method comprising: (a) expanding mammalian cells thatsecrete a recombinant glycoprotein, wherein the expanding is from a seedculture to a liquid culture of at least about 10,000 L, wherein therecombinant protein concentration is at least about 0.5 g/L of liquidculture; and (b) isolating the recombinant protein from the at leastabout 10,000 L liquid culture, wherein the isolating comprises: (i)obtaining a soluble fraction of the culture of step (a); (ii) subjectingthe soluble fraction to affinity chromotagraphy to obtain an elutedprotein product; (iii) subjecting the eluted protein product of step(ii) to anion exchange chromatography so as to obtain an eluted andenriched protein product; and (iv) subjecting the enriched proteinproduct to hydrophobic interaction chromatography to obtain an enrichedprotein product.

In one embodiment of the invention, the protein comprises a CTLA4-Ig. Inanother embodiment, the protein comprises a beta polypeptide or betapolypeptide molecules. In another embodiment, the protein comprises betapolypeptides having SEQ ID NO: 11, 12, 13, 14, 15, or 16.

In one embodiment of the invention, the at least four passagescomprises: (i) growing the cells in a culture volume of at least 50 mLuntil a cell density of from about 1 million to about 2.5 million cellsper ml is reached; (ii) growing the cells in a culture volume of atleast 10 L until a cell density of about 1 million to about 2.5 millioncells per ml is reached; (iii) growing the cells in a culture volume ofat least 100 L until a cell density of about 1 million to about 2.5million cells per ml is reached; and (iv) growing the cells in a culturevolume of 200 L until a cell density of about 1 million to about 2.5million cells per ml is reached.

In one embodiment of the invention, the isolating comprises: (i)obtaining a soluble fraction of the culture of step (a); (ii) subjectingthe soluble fraction to affinity chromotagraphy to obtain an elutedprotein product; (iii) subjecting the eluted protein product of step(ii) to anion exchange chromatography so as to obtain an eluted andenriched protein product; and (iv) subjecting the enriched proteinproduct to hydrophobic interaction chromatography to obtain an enrichedprotein product.

In one embodiment, the enriched protein product obtained in step (iv) ischaracterized in that a percentage of any high molecular weight multimeris less than 25% by weight. In another embodiment, the anion exchangechromatography of step (iii) is carried out using a wash buffercomprising about 50 mM HEPES, and about 135 mM NaCl, and having a pH ofabout 7. In another embodiment, the anion exchange chromatography ofstep (iii) is carried out using an elution buffer comprising about 50 mMHEPES, and about 200 mM NaCl, and having a pH of about 7. In anotherembodiment, the hydrophobic interaction chromatography of step (iv) iscarried out using a wash buffer comprising about 50 mM HEPES, and about1.2 M (NH₄)₂SO₄, and having a pH of about 7. In another embodiment, theaffinity chromatography of step (ii) is carried out using a wash buffercomprising about 25 mM NaH₂PO₄, and about 150 mM NaCl, and having a pHof about 7.5. In another embodiment, the affinity chromatography of step(ii) is carried out using an elution buffer comprising about 250 mMGlycine and having a pH of about 3. In another embodiment, the anionexchange chromatography of step (iii) is carried out using a columnhaving an anion exchange resin having a primary, secondary, tertiary, orquartenary amine functional group. In another embodiment, the resin hasa quarternary amine functional group. In another embodiment, thehydrophobic interaction chromatography of step (iii) is carried outusing a hydrophobic interaction resin having a phenyl, an octyl, apropyl, an alkoxy, a butyl, or an isoamyl functional group. In anotherembodiment, the functional group is a phenyl functional group. Inanother embodiment, the affinity chromatography of step (ii) is carriedout using a column containing Protein A.

In another embodiment, the expanding comprises: (i) culturing the cellsin a serum-free, chemically defined medium with at least four passagesso as to obtain a cell density of at least about 1.0×10⁵ viable cellsper mL, wherein each seed stage starts at about 2×10⁵ per ml and goes toabout 1-2 million cells per ml; and (ii) maintaining the cells inculture for a time sufficient to produce the recombinant protein fromthe culture at least about 0.5 g/L. In another embodiment, the culturingcomprises: (i) culturing the cells in a serum-free, chemically-definedinoculum medium for from about 15 days to about 25 days; then (ii)culturing the cells in a serum-free, chemically-defined basal mediumuntil a cell density of about at least 4 million cells per mL isreached.

In another embodiment, the maintaining comprises (i) lowering thetemperature of the culture from 37±2° C. to 34±2° C.; and (ii) adding apolyanionic compound to the culture. In one embodiment, the polyanioniccompound is dextran sulfate and wherein the dextran sulfate is added tothe culture at a final concentration of about 50 mg/ml. In anotherembodiment, the temperature is kept within the range of 34±2° C. for atleast 5 days. In another embodiment, the temperature is kept within therange of 34±2° C. for at least 6 days. In another embodiment, thetemperature is kept within the range of 34±2° C. for at least 7 days. Inanother embodiment, the temperature is kept within the range of 34±2° C.for at least 8 days. In another embodiment, the temperature is keptwithin the range of 34±2° C. for at least 9 days. In another embodiment,the temperature is kept within the range of 34±2° C. for at least 10days. In another embodiment, the temperature is kept within the range of34±2° C. for at least 11 days. In another embodiment, the temperature iskept within the range of 34±2° C. for at least 12 days. In anotherembodiment, the temperature is kept within the range of 34±2° C. for atleast 13 days. In another embodiment, the temperature is kept within therange of 34±2° C. for at least 14 days. In another embodiment, thetemperature is kept within the range of 34±2° C. for at least 15 days.In another embodiment, the temperature is kept within the range of 34±2°C. for at least 16 days. In another embodiment, the temperature is keptwithin the range of 34±2° C. for at least 17 days. In anotherembodiment, the temperature is kept within the range of 34±2° C. for atleast 18 days. In another embodiment, the temperature is kept within therange of 34±2° C. for at least 19 days. In another embodiment, thetemperature is kept within the range of 34±2° C. for at least 20 days.In another embodiment, the temperature is kept within the range of 34±2°C. for at least 21 days. In another embodiment, the temperature is keptwithin the range of 34±2° C. for at least 22 days. In anotherembodiment, the temperature is kept within the range of 34±2° C. for atleast 23 days. In another embodiment, the temperature is kept within therange of 34±2° C. for at least 24 days. In another embodiment, thetemperature is kept within the range of 34±2° C. for at least 25 days.In another embodiment, the temperature is kept within the range of 34±2°C. for at least 26 days. In another embodiment, the temperature is keptwithin the range of 34±2° C. for at least 27 days. In anotherembodiment, the temperature is kept within the range of 34±2° C. for atleast 28 days. In another embodiment, the temperature is kept within therange of 34±2° C. for up to 28 days. In another embodiment, thetemperature is kept within the range of 34±2° C. until the cell densityof the culture is from about 30×10⁵ to about 79×10⁵ cells per mL ofliquid culture.

In one embodiment, the time sufficient is a time by which the cells'viability does not fall below 30%. In another embodiment, the timesufficient is a time by which the cells' viability does not fall below40%. In another embodiment, the time sufficient is a time by which thecells' viability does not fall below 50%. In another embodiment, thetime sufficient is a time by which the cells' viability does not fallbelow 60%. In another embodiment, the time sufficient is a time by whichthe cells' viability does not fall below 70%, or 80% or 90% or 95%.

In one embodiment, galactose is added to the serum-free, chemicallydefined medium. In another embodiment, isolating occurs when the liquidculture contains greater than or equal to about 6 moles of sialic acidper mole of protein. In another embodiment, isolating occurs when theliquid culture contains from about 5.2 to about 7.6 moles of sialic acidper mole of protein. In another embodiment, the isolating occurs whenthe liquid culture has a cell density of from about 33×10⁵ to about79×10⁵ cells per mL. In another embodiment, the isolating occurs whencell viability in the liquid culture has not fallen below about 37%. Inanother embodiment, the isolating occurs when endotoxin is less than orequal to about 4.8 EU per mL of liquid culture. In another embodiment,the isolating occurs when bioburden is less than about 1 colony formingunit per mL (cfu/ml) of liquid culture. In another embodiment, theisolating occurs if at least two of the following conditions are met:(i) the liquid culture contains greater than or equal to about 6 molesof sialic acid per mole of protein, (ii) the liquid culture has a celldensity of from about 33×10⁵ to about 79×10⁵ cells per mL, (iii) cellviability in the liquid culture has not fallen below about 37%, or (iv)the amount of glycoprotein in the culture is from about 0.46 g/L toabout 0.71 g/L.

In one embodiment, the mammalian cells are progeny of a Chinese HamsterOvary clonal cell line that produces any combination of betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises SEQ ID NO: 11, 12, 13, 14, 15, or 16, wherein the ChineseHamster Ovary cells each have stably integrated in their genome at least30 copies of an expression cassette comprising SEQ ID NO:3. In oneembodiment, the liquid culture comprises a cell of or a progeny cell ofa cell a production cell line of the invention.

The invention provides for a beta polypeptide comprising SEQ ID NO: 11,12, 13, 14, 15, or 16 obtained by a method provided by the invention.The invention provides for a composition comprising beta polypeptides orbeta polypeptide molecules, wherein each polypeptide comprises SEQ IDNO: 11, 12, 13, 14, 15, or 16 obtained by a method provided by theinvention. The invention provides for a beta polypeptide obtained by amethod provided by the invention.

Cells: The invention provides for a clonal Chinese Hamster Ovary cellcomprising a nucleic acid encoding a beta polypeptide or a betapolypeptide molecule. The invention provides for a clonal ChineseHamster Ovary cell population that produces beta polypeptides or betapolypeptide molecules. In one embodiment, the beta polypeptide comprisesSEQ ID NO: 11, 12, 13, 14, 15, or 16. The invention provides for aclonal Chinese Hamster Ovary cell comprising a nucleic acid comprisingan expression cassette encoding the amino acid sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16. In one embodiment, the expression cassettecomprises SEQ ID NO:3. The invention provides for a clonal ChineseHamster Ovary cell population that produces a beta polypeptide or betapolypeptide molecule, wherein the beta polypeptide is expressed from anucleotide sequence derived from a plasmid having ATCC Accession No.PTA-2104 deposited under the provisions of the Budapest Treaty on June19, 2000 with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va., 20110.

The invention provides for a clonal Chinese Hamster Ovary cellpopulation that produces a beta polypeptide or beta polypeptidemolecules, each cell comprising 30 or more copies of a beta polypeptideexpression cassette. The invention provides for a clonal Chinese HamsterOvary cell population that produces a beta polypeptide or betapolypeptide molecules, each cell comprising 30 or more copies of a betapolypeptide expression cassette, wherein the 30 or more copies areintegrated at a single site in the genome of each cell. The inventionprovides for a clonal Chinese Hamster Ovary cell population thatproduces a beta polypeptide or beta polypeptide molecules, wherein abeta polypeptide expression cassette is stable over about 105 passages.In one embodiment, the beta polypeptide is encoded by an expressioncassette integrated into a cell genome.

The invention provides for a clonal Chinese Hamster Ovary cellpopulation that produces a beta polypeptide, wherein at least 75% of thepopulation of cells has 30 or more copies of a beta polypeptideexpression cassette per cell, wherein the 30 or more copies areintegrated at a single site in the genome of each cell of the 75% of thepopulation. The invention provides for a clonal Chinese Hamster Ovarycell population that produces a beta polypeptide, wherein at least 85%of the population of cells has 30 or more copies of a beta polypeptideexpression cassette per cell, wherein the 30 or more copies areintegrated at a single site in the genome of each cell of the 85% of thepopulation. The invention provides for a clonal Chinese Hamster Ovarycell population that produces a beta polypeptide, wherein at least 95%of the population of cells has 30 or more copies of a beta polypeptideexpression cassette per cell, wherein the 30 or more copies areintegrated at a single site in the genome of each cell of the 95% of thepopulation. In one embodiment, the expression cassette is derived from aplasmid deposited as ATCC Accession No. PTA-2104. In another embodiment,the expression cassette comprises a nucleic acid having the sequence ofSEQ ID NO:3. In one embodiment, the cell population produces at leastabout 0.5 grams of the beta polypeptide per liter of liquid culture, andwherein the beta polypeptide has a molar ratio of sialic acid to betapolypeptide dimer or beta polypeptide molecule of from about 5.5 toabout 8.5 at a culture scale of 1,000 L or more. In another embodiment,the cell population produces at least 5, at least 10 or at least 20grams of the beta polypeptide per liter of liquid culture. In anotherembodiment, the beta polypeptide has a molar ratio of sialic acid tobeta polypeptide dimer or beta polypeptide molecule of from about 5 toabout 10 at a culture scale of 1,000 L or more. In another embodiment,the cell population has been adapted to a serum-free, chemically definedmedium. In another embodiment, a beta polypeptide produced from cultureof the cell population has an extinction coefficient of 1.0±0.05 AU mLcm⁻¹ mg⁻¹. In another embodiment, the cell population, when grown inculture, produces beta polypeptides, wherein: (a) about 90% or about 80%of the beta polypeptides comprise an amino acid sequence of SEQ ID NO:4beginning with the methionine at residue 27; (b) about 10% or about 20%of the beta polypeptides comprise the amino acid sequence of SEQ ID NO:4beginning with the alanine at residue number 26; (c) from about 4% toabout 8% of the beta polypeptides comprise the amino acid sequence ofSEQ ID NO:4 ending with the lysine at residue number 383; (d) from about92% to about 96% of the beta polypeptides comprise the amino acidsequence of SEQ ID NO:4 ending with the glycine at residue number 382;and optionally, (e) about less than 1% of the beta polypeptides comprisethe amino acid sequence of SEQ ID NO:4 beginning with the methionine atresidue number 25.

The invention provides for a progeny cell of a cell population of theinvention, wherein the progeny cell produces a beta polypeptide. In oneembodiment, the progeny cell is obtained from culturing a cell over atleast 5, at least 10, at least 20, at least 40, at least 50, at least 75generations. In another embodiment, the progeny cell is obtained fromculturing a cell for 27 generations.

The invention provides for a cell line produced from any cell providedby the invention. In one embodiment, the cell line is clonal. In oneembodiment, the cell line produces: (a) a beta polypeptide having anamino acid sequence of SEQ ID NO:16 (methionine at amino acid position27 and glycine at amino acid position 382 of SEQ ID NO:4); (b) a betapolypeptide having an amino acid sequence of SEQ ID NO:13 (methionine atamino acid position 27 and lysine at amino acid position 383 of SEQ IDNO:4); (c) a beta polypeptide having an amino acid sequence of SEQ IDNO: 15 (alanine at amino acid position 26 and glycine at amino acidposition 382 of SEQ ID NO:4); (d) a beta polypeptide having an aminoacid sequence of SEQ ID NO: 12 (alanine at amino acid position 26 andlysine at amino acid position 383 of SEQ ID NO:4); (e) a betapolypeptide having an amino acid sequence of SEQ ID NO: 11 (methionineat amino acid position 25 and lysine at amino acid position 383 of SEQID NO:4); (f) a beta polypeptide having an amino acid sequence of SEQ IDNO: 14 (methionine at amino acid position 25 and glycine at amino acidposition 382 of SEQ ID NO:4); or (g) any combination thereof. In oneembodiment, the cell line produces beta polypeptides or beta polypeptidemolecules, wherein: (a) about 90% or about 80% of the beta polypeptidescomprise an amino acid sequence of SEQ ID NO:4 beginning with themethionine at residue 27; (b) about 10% or about 20% of the betapolypeptides comprise the amino acid sequence of SEQ ID NO:4 beginningwith the alanine at residue number 26; (c) from about 4% to about 8% ofthe beta polypeptides comprise the amino acid sequence of SEQ ID NO:4ending with the lysine at residue number 383; (d) from about 92% toabout 96% of the beta polypeptides comprise the amino acid sequence ofSEQ ID NO:4 ending with the glycine at residue number 382; andoptionally, (e) about less than 1% of the beta polypeptides comprise theamino acid sequence of SEQ ID NO:2 beginning with the methionine atresidue number 25. In one embodiment, wherein the beta polypeptides,which are produced from culturing the cell line, have an extinctioncoefficient of 1.0±0.05 AU mL cm-1 mg-1.

The invention provides for a cell population derived from a cell of theinvention. In one embodiment, the cells of the population contain atleast one additional genetic change as compared to the cell of theinvention from which the population was derived, and wherein the cellsproduce a beta polypeptide. In another embodiment, the cells of thepopulation contain at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, or at least 20additional genetic change as compared to the cell of the invention fromwhich the population was derived, and wherein the cells produce a betapolypeptide. In one embodiment, the genetic change comprises at leastone non-conservative mutation in the cellular genome or in theexpression cassette encoding the beta polypeptide. In anotherembodiment, the genetic change comprises at least one additionalrecombinant nucleic acid within the cell. In another embodiment, thegenetic change comprises a mutation of the cellular genome. In anotherembodiment, the genetic change comprises addition of a nucleic acid tothe cell genome or as a trans nucleic acid, and wherein the nucleic acidencodes an anti-apoptotic polypeptide. In another embodiment, theanti-apoptotic polypeptide relates to glycosylation. In anotherembodiment, the genetic change comprises at least one mutation of thecellular genome or of the expression cassette encoding a betapolypeptide. In another embodiment, the cell population, when grown inculture, produces: (a) a beta polypeptide having an amino acid sequenceof SEQ ID NO:16 (methionine at amino acid position 27 and glycine atamino acid position 382 of SEQ ID NO:4); (b) a beta polypeptide havingan amino acid sequence of SEQ ID NO:7 (methionine at amino acid position27 and lysine at amino acid position 383 of SEQ ID NO:4); (c) a betapolypeptide having an amino acid sequence of SEQ ID NO:15 (alanine atamino acid position 26 and glycine at amino acid position 382 of SEQ IDNO:4); (d) a beta polypeptide having an amino acid sequence of SEQ IDNO:12 (alanine at amino acid position 26 and lysine at amino acidposition 383 of SEQ ID NO:4); (e) a beta polypeptide having an aminoacid sequence of SEQ ID NO:11 (methionine at amino acid position 25 andlysine at amino acid position 383 of SEQ ID NO:4); (f) a betapolypeptide having an amino acid sequence of SEQ ID NO:14 (methionine atamino acid position 25 and glycine at amino acid position 382 of SEQ IDNO:4); or (g) any combination thereof.

Compositions: The invention provides for an isolated population of betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, havingan average molar ratio of sialic acid groups to beta polypeptide dimeror beta polypeptide molecule of from about 5 to about 10. In oneembodiment, the average molar ratio of sialic acid groups to betapolypeptide dimer or beta polypeptide molecule of from about 5.5 toabout 8.5. In one embodiment, average molar ratio of sialic acid groupsto beta polypeptide dimer or beta polypeptide molecule of from about 5.2to about 7.6. The invention provides for an isolated population of betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, having an average molar ratio of sialicacid groups to beta polypeptide dimer or beta polypeptide molecule ofabout 6. The invention provides for an isolated population of betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDN011, 12, 13, 14, 15, or 16, and wherein greater than 95% of thepolypeptides are formed into dimers. In one greater than 98%, greaterthan 99%, or greater than 99.5% of the polypeptides are formed intodimers. In another embodiment, from about 95% to about 99.5% of thepolypeptides are formed into dimers and about 0.5% to about 5% of thepolypeptides are formed into tetramers or high molecular weight species.In another embodiment, about 98.6% of the polypeptides are formed intodimers and about 1.2% of the polypeptides are formed into tetramers orhigh molecular weight species and about less than 0.7% of thepolypeptides are monomers. In another embodiment, about 95% of thepolypeptides are formed into dimers and about 4% of the polypeptides areformed into tetramers or high molecular weight species and about 1% ofthe polypeptides are isolated population of beta polypeptide dimers,wherein each polypeptide monomer comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16. In one embodiment, the population issubstantially free of beta polypeptide monomer. In another embodiment,the population is substantially free of beta polypeptide tetramer. Theinvention provides for an isolated population of beta polypeptidemonomers substantially free of beta polypeptide dimer and tetramer. Inone embodiment, each monomer of each beta polypeptide dimer comprisesthe sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16and has at least 2.5sialic acid groups.

The invention provides for an isolated population of beta polypeptides,wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13, 14, 15, or 16, having a potency of from about 70% to about 130% in aB7 binding assay, compared to a CTLA4-Ig standard, wherein the assaycomprises measuring surface plasmon resonance. The invention providesfor an isolated population of beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, having a potency of from about 50% to about 150% in a human cellIL-2 inhibition assay, compared to a standard. The invention providesfor a purified population of beta polypeptide tetramers or highmolecular weight species, wherein each polypeptide monomer comprises thesequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, the population beingsubstantially free of beta polypeptide dimers, and optionally whereinthe population comprises an amount that is greater than about 100 grams.The invention provides for a purified population of beta polypeptidetetramers or high molecular weight species, wherein each polypeptidemonomer comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16,the population being substantially free of beta polypeptide monomer, andoptionally wherein the population comprises an amount that is greaterthan about 100 grams. In one embodiment, each tetramer moleculecomprises two pairs of beta polypeptides, wherein each polypeptidemonomer comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16,and wherein each member of the pair of polypeptides is covalently linkedto the other member, and wherein the two pairs of polypeptides arenon-covalently associated with one another. In one embodiment, eachtetramer molecule is capable of binding to a CD80 or CD86. In oneembodiment, each tetramer molecule has at least a 2-fold greater avidityfor CD80 or CD86 as compared to a beta polypeptide dimer, wherein eachpolypeptide monomer of the dimer comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16. In another embodiment, each tetramer moleculehas at least a 2-fold greater avidity for CD80 or CD86 as compared to aCTLA4-Ig tetramer molecule comprising the sequence of SEQ ID NO:2. Inanother embodiment, each tetramer molecule has at least a 2-fold greaterinhibition of T cell proliferation or activation as compared to a betapolypeptide dimer, wherein each polypeptide monomer of the dimercomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16. Inanother embodiment, each tetramer molecule has at least a 2-fold greaterinhibition of T cell proliferation or activation as compared to aCTLA4-Ig tetramer molecule comprising the sequence of SEQ ID NO:2.

The invention provides for an isolated composition comprising betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the composition comprises dominant isoforms visualizable on anisoelectric focusing gel which have an isoelectric point, pI, less thanor equal to 5.5 as determined by isoelectric focusing. In oneembodiment, the pI increases after neuraminidase treatment. In oneembodiment, at least 40% of the beta polypeptides or beta polypeptidemolecules exhibit an isoelectric point less than or equal to about 5.3as determined by isoelectric focusing. In one embodiment, at least 70%of the beta polypeptides or beta polypeptide molecules exhibit anisoelectric point less than or equal to about 5.3 as determined byisoelectric focusing. In one embodiment, at least 90% of the betapolypeptides or beta polypeptide molecules exhibit an isoelectric pointless than or equal to about 5.3 as determined by isoelectric focusing.The invention provides for an isolated population of beta polypeptidesor beta polypeptide molecules having a pI of from about 2.0±0.2 to about5.2±0.2. The invention provides for an isolated population of betapolypeptides or beta polypeptide molecules having a pI from about4.5±0.2 to about 5.2±0.2. The invention provides for an isolatedpopulation of beta polypeptides or beta polypeptide molecules having apI of about 4.7±0.2 to about 5.1±0.2. The invention provides for amethod for preparing a composition, the composition comprising betapolypeptides or beta polypeptide molecules with a pI of from about2.0±0.2 to about 5.2±0.2, wherein each polypeptide comprises thesequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, the method comprising:(a) subjecting a mixture of beta polypeptides to isoelectric focusinggel electrophoresis, wherein a single band on the gel represents apopulation of beta polypeptides or beta polypeptide molecules with aparticular pI, and (b) isolating the population of beta polypeptides orbeta polypeptide molecules having a pI of from about 2.0±0.2 to about5.2±0.2 so as to prepare the composition.

The invention provides for an isolated composition comprising betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the polypeptides are characterized by an average molar ratio ofGlcNAc per mole of beta polypeptide dimer or beta polypeptide moleculeof from about 24 to about 28. The invention provides for an isolatedcomposition comprising beta polypeptides, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the polypeptides are characterized by an average molar ratio ofGalNAc per mole of beta polypeptide dimer or beta polypeptide moleculeof from about 2.7 to about 3.6. The invention provides for an isolatedcomposition comprising beta polypeptides, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the polypeptides are characterized by an average molar ratio ofgalactose per mole of beta polypeptide dimer or beta polypeptidemolecule of from about 11 to about 13. The invention provides for anisolated composition comprising beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, and wherein the polypeptides are characterized by an average molarratio of fucose per mole of beta polypeptide dimer or beta polypeptidemolecule of from about 6.4 to about 7.0. The invention provides for anisolated composition comprising beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, and wherein the polypeptides are characterized by an average molarratio of mannose per mole of beta polypeptide dimer or beta polypeptidemolecule of from about 14 to about 16. The invention provides for anisolated composition comprising beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, and wherein the molecules are characterized by an average molarratio of sialic acid per mole of beta polypeptide dimer or betapolypeptide molecule of from about 5.5 to about 8.5. The inventionprovides for an isolated composition comprising beta polypeptides,wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13, 14, 15, or 16, and wherein the molecules are characterized by anaverage molar ratio of sialic acid per mole of beta polypeptide dimer orbeta polypeptide molecule of from about 5 to about 10. The inventionprovides for an isolated composition comprising beta polypeptides,wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13, 14, 15, or 16, and wherein the polypeptides are characterized by:(a) an average molar ratio of GlcNAc per mole of beta polypeptide dimeror beta polypeptide molecule from about 24 to about 28; and (b) anaverage molar ratio of sialic acid per mole of beta polypeptide dimer orbeta polypeptide molecule from about 5.5 to about 8.5. The inventionprovides for an isolated composition comprising beta polypeptides,wherein each polypeptide comprises the sequence of SEQ ID NO: 11, 12,13, 14, 15, or 16, and wherein the molecules are characterized by: (a)an average molar ratio of GlcNAc per mole of beta polypeptide dimer orbeta polypeptide molecule from about 24 to about 28; (b) an averagemolar ratio of GalNAc per mole of beta polypeptide dimer or betapolypeptide molecule from about 2.7 to about 3.6; and (c) an averagemolar ratio of sialic acid per mole of beta polypeptide dimer or betapolypeptide molecule from about 5.5 to about 8.5. The invention providesfor an isolated composition comprising beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, and wherein the molecules are characterized by: (a) an average molarratio of GlcNAc per mole of beta polypeptide dimer or beta polypeptidemolecule from about 24 to about 28; (b) an average molar ratio of GalNAcper mole of beta polypeptide dimer or beta polypeptide molecule fromabout 2.7 to about 3.6; (c) an average molar ratio of galactose per moleof beta polypeptide dimer or beta polypeptide molecule from about 11 toabout 13; and (d) an average molar ratio of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule from about 5.5 to about8.5. The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of GlcNAc per mole of betapolypeptide dimer or beta polypeptide molecule from about 24 to about28; (b) an average molar ratio of GalNAc per mole of beta polypeptidedimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) anaverage molar ratio of galactose per mole of beta polypeptide dimer orbeta polypeptide molecule from about 11 to about 13; (d) an averagemolar ratio of fucose per mole of beta polypeptide dimer or betapolypeptide molecule from about 6.4 to about 7.0; and (e) an averagemolar ratio of sialic acid per mole of beta polypeptide dimer or betapolypeptide molecule from about 5.5 to about 8.5. The invention providesfor an isolated composition comprising beta polypeptides, wherein eachpolypeptide comprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or16, and wherein the polypeptides are characterized by: (a) an averagemolar ratio of GlcNAc per mole of beta polypeptide dimer or betapolypeptide molecule from about 24 to about 28; (b) an average molarratio of GalNAc per mole of beta polypeptide dimer or beta polypeptidemolecule from about 2.7 to about 3.6; (c) an average molar ratio ofgalactose per mole of beta polypeptide dimer or beta polypeptidemolecule from about 11 to about 13; (d) an average molar ratio of fucoseper mole of beta polypeptide dimer or beta polypeptide molecule fromabout 6.4 to about 7.0; (e) an average molar ratio of mannose per moleof beta polypeptide dimer or beta polypeptide molecule from about 14 toabout 16; and (f) an average molar ratio of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule from about 5.5 to about8.5. The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of galactose per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 8 toabout 17; (b) an average molar ratio of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule from about 5.5 to about8.5; and (c) a carbohydrate profile substantially the same as FIG. 8.The invention provides for an isolated composition comprising betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the polypeptides are characterized by: (a) an average molarratio of galactose per mole of beta polypeptide dimer or betapolypeptide molecule from about 8 to about 17; (b) an average molarratio of sialic acid per mole of beta polypeptide dimer or betapolypeptide molecule from about 5.5 to about 8.5; (c) a carbohydrateprofile substantially the same as FIG. 8; and (d) a beta polypeptidetetramer content less than about 5%. The invention provides for anisolated composition comprising beta polypeptides or beta polypeptidemolecules, wherein each polypeptide comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of galactose per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 11 toabout 13; and (b) an average molar ratio of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule from about 5.5 to about8.5. The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of galactose per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 11 toabout 13; (b) an average molar ratio of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule from about 5.5 to about8.5; and (c) a beta polypeptide tetramer content less than about 5%. Theinvention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein polypeptides arecharacterized by: (a) an average molar ratio of sialic acid per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 5.5 toabout 8.5; and (b) a carbohydrate profile substantially the same as FIG.8. The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of galactose per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 11 toabout 13; and (b) a carbohydrate profile substantially the same as FIG.8. The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein polypeptides arecharacterized by: (a) an average molar ratio of sialic acid per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 5.5 toabout 8.5; and (b) a beta polypeptide tetramer or high molecular weightspecies content less than about 5%. The invention provides for anisolated composition comprising beta polypeptides or beta polypeptidemolecules, wherein each polypeptide comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16, and wherein the polypeptides arecharacterized by: (a) an average molar ratio of galactose per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 11 toabout 13; and (b) a beta polypeptide tetramer or high molecular weightspecies content less than about 5%.

The invention provides for an isolated composition comprising betapolypeptides or beta polypeptide molecules, wherein each polypeptidecomprises the sequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, andwherein the polypeptides exhibit a carbohydrate profile substantiallythe same as FIG. 8. The invention provides for an isolated compositioncomprising beta polypeptides, wherein each polypeptide comprises thesequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein thepolypeptides exhibit a carbohydrate profile of Domains I-IV, whereinDomain I comprises peaks which represent a-sialylated oligosaccharides,Domain II comprises peaks which represent mono-sialylatedoligosaccharides, Domain III comprises peaks which representdi-sialylated oligosaccharides, and Domain IV comprises peaks whichrepresent tri-sialylated oligosaccharides. In one embodiment, thedifference in retention times of N-linked oligosaccharides between afirst peak in Domain I and a main peak in Domain II is from about 11 toabout 13 minutes. In one embodiment, the sum of Domains III and IVcomprises from about 25% to about 36% of the total carbohydrate profile.

The invention provides for an isolated composition comprising betapolypeptide dimers or beta polypeptide molecules, wherein eachpolypeptide monomer comprises the sequence of SEQ ID NO: 11, 12, 13, 14,15, or 16, and wherein at least about 0.5% of the molecules arecysteinylated. The invention provides for an isolated population of betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the population exhibits amass spectrometry profile as shown in FIG. 10. The invention providesfor an isolated population of beta polypeptides or beta polypeptidemolecules, wherein each polypeptide comprises the sequence of SEQ ID NO:11, 12, 13, 14, 15, or 16, having an average molar ratio of sialic acidgroups to beta polypeptide dimer or beta polypeptide molecule of fromabout 5.5 to about 8.5, wherein the beta polypeptide dimer or betapolypeptide molecules is produced from cells of a production cell line.The invention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, wherein the polypeptides are glycosylatedat an asparagine amino acid residue at position 102 of SEQ ID NO:4, anasparagine amino acid residue at position 134 of SEQ ID NO:4, anasparagine amino acid residue at position 233 of SEQ ID NO:4. Theinvention provides for an isolated composition comprising betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the molecules arecharacterized by: (a) an average molar ratio of GlcNAc per mole of betapolypeptide dimer or beta polypeptide molecule from about 24 to about28; (b) an average molar ratio of GalNAc per mole of beta polypeptidedimer or beta polypeptide molecule from about 2.7 to about 3.6; (c) anaverage molar ratio of galactose per mole of beta polypeptide dimer orbeta polypeptide molecule from about 11 to about 13; (d) an averagemolar ratio of fucose per mole of beta polypeptide dimer or betapolypeptide molecule from about 6.4 to about 7.0; (e) an average molarratio of mannose per mole of beta polypeptide dimer or beta polypeptidemolecule from about 14 to about 16; (f) an average molar ratio of sialicacid per mole of beta polypeptide dimer or beta polypeptide moleculefrom about 5.5 to about 8.5; (g) a pI as determined from visualizationon an isoelectric focusing gel in a range from about 2.4±0.2 to about5.2±0.2; (h) MCP-1 of less than or equal to 5 ppm; (i) less than 5%tetramer or high molecular weight species; (j) less than betapolypeptide 1% monomer; and (k) beta polypeptides or beta polypeptidemolecules of the population having an amino acid at least 95% identicalto any of SEQ ID NOS:4, 11, 12, 13, 14, 15, or 16, wherein the betapolypeptides within the population are capable of binding to CD80 andCD86. The invention provides for an isolated population of betapolypeptides, wherein each polypeptide comprises the sequence of SEQ IDNO: 11, 12, 13, 14, 15, or 16, and wherein the population of moleculesis characterized by: (a) an average molar ratio of GlcNAc per mole ofbeta polypeptide dimer or beta polypeptide molecule from about 24 toabout 28; (b) an average molar ratio of GalNAc per mole of betapolypeptide dimer or beta polypeptide molecule from about 2.7 to about3.6; (c) an average molar ratio of galactose per mole of betapolypeptide dimer or beta polypeptide molecule from about 11 to about13; (d) an average molar ratio of fucose per mole of beta polypeptidedimer or beta polypeptide molecule from about 6.4 to about 7.0; (e) anaverage molar ratio of mannose per mole of beta polypeptide dimer orbeta polypeptide molecule from about 14 to about 16; (f) an averagemolar ratio of sialic acid per mole of beta polypeptide dimer or betapolypeptide molecule from about 5.5 to about 8.5; (g) a pI as determinedfrom visualization on an isoelectric focusing gel in a range from about2.4±0.2 to about 5.2±0.2; (h) MCP-1 of less than or equal to 5 ppm; (i)less than 5% beta polypeptide tetramer or high molecular weight; (j)less than 1% monomer; and (k) beta polypeptides of the population havingan amino acid at least 95% identical to any of SEQ ID NOS:4, 11, 12, 13,14, 15, or 16, wherein beta polypeptide molecules within the populationare capable of binding to CD80 and CD86; or pharmaceutical equivalentsthereof

The invention provides for a composition comprising an effective amountof the beta polypeptide of the invention and a pharmaceuticallyacceptable carrier. The invention provides for a composition comprisingexcipients as described in U.S. Application No. 60/752,150; filed Dec.20, 2005. In one embodiment, the composition includes beta polypeptidemolecules. In one embodiment, the composition further comprises apharmaceutically acceptable diluent, adjuvant or carrier. In oneembodiment, the composition further comprises sucrose, sodium phosphatemonobasic monohydrate, sodium chloride, sodium hydroxide, hydrochloricacid, and sterile water. In another embodiment, the compositioncomprises sucrose, poloxamer, sodium phosphate monobasic monohydrate,sodium phosphate dibasic anhydrous, sodium chloride, sodium hydroxide,and sterile water. In one embodiment, the composition is lyophilized.The invention provides for a lyophilized composition comprising aneffective amount of the beta polypeptides of the invention, sucrose,sodium phosphate monobasic monohydrate, sodium chloride, sodiumhydroxide, and hydrochloric acid.

Formulations and kits: The invention provides for lyophilized betapolypeptide mixture, wherein each polypeptide comprises the sequence ofSEQ ID NO: 11, 12, 13, 14, 15, or 16, comprising at least 95% betapolypeptide dimer, and not more than 5% beta polypeptide tetramer (highmolecular weight species). In one embodiment, the mixture comprises atleast 98% beta polypeptide dimer and no more than 2% beta polypeptidetetramer (high molecular weight species). In one embodiment, the mixturecomprises at least 99% beta polypeptide dimer and no more than 1% betapolypeptide tetramer (high molecular weight species). In one embodiment,the mixture comprises at least 5 moles of sialic acid per mole of betapolypeptide dimer or beta polypeptide molecule. In one embodiment, themixture comprises from about 24 to about 28 moles of GlcNAc per mole ofbeta polypeptide dimer (high molecular weight species). In oneembodiment, the mixture comprises from about 2.7 to about 3.6 moles ofGalNAc per mole of beta polypeptide dimer or beta polypeptide molecule.In one embodiment, the mixture comprises from about 11 to about 13 molesof galactose per mole of beta polypeptide dimer or beta polypeptidemolecule. In one embodiment, the mixture comprises from about 6.4 toabout 7.0 moles of fucose per mole of beta polypeptide dimer or betapolypeptide molecule. In one embodiment, the mixture comprises fromabout 14 to about 16 moles of mannose per mole of beta polypeptide dimeror beta polypeptide molecule. The invention also provides for apharmaceutical kit comprising: (a) a container containing a lyophilizedbeta polypeptide mixture of the invention and (b) instructions forreconstituting the lyophilized beta polypeptide mixture into solutionfor injection.

Illustrative methods of treatment: A method for inhibiting T cellproliferation, activation or both, the method comprising contacting a Tcell with an effective amount of a beta polypeptide composition of theinvention. The invention provides for a method for inhibiting an immuneresponse in a subject, the method comprising administering to a subjectin need thereof an effective amount of a beta polypeptide composition ofthe invention. The invention provides for a method for treating animmune disorder in a subject, the method comprising administering to asubject in need thereof an effective amount of a beta polypeptidecomposition of the invention. The invention provides for a method forinducing immune tolerance to an antigen in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The methodprovides for a method for treating inflammation in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The methodprovides for a method for treating rheumatoid arthritis comprisingadministering to a subject in need thereof an effective amount of a betapolypeptide composition of the invention. The invention provides for amethod for treating psoriasis in a subject, the method comprisingadministering to a subject in need thereof an effective amount of a betapolypeptide composition of the invention. The invention provides for amethod for treating lupus in a subject, the method comprisingadministering to a subject in need thereof an effective amount of a betapolypeptide composition of the invention. The invention provides for amethod for treating or preventing an allergy in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The inventionprovides for a method for treating or preventing graft versus hostdisease in a subject, the method comprising administering to a subjectin need thereof an effective amount of a beta polypeptide composition ofthe invention. The invention provides for a method for treating orpreventing rejection of a transplanted organ in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The inventionprovides for a method for treating or preventing rejection oftransplanted tissue in a subject, the method comprising administering toa subject in need thereof an effective amount of the composition a betapolypeptide composition of the invention. The invention provides for amethod for treating or preventing rejection of a transplanted cell in asubject, the method comprising administering to a subject in needthereof an effective amount of a beta polypeptide composition of theinvention. In one embodiment, the transplanted cell is a bone marrowcell. In another embodiment, the transplanted cell is an islet cell. Inanother embodiment, the transplanted cell is an insulin-producingpancreatic islet cell. The invention provides for a method for treatingmultiple sclerosis in a subject, the method comprising administering toa subject in need thereof an effective amount of a beta polypeptidecomposition of the invention. The invention provides for a method fortreating Crohn's Disease in a subject, the method comprisingadministering to a subject in need thereof an effective amount of a betapolypeptide composition of the invention. The invention provides for amethod for treating type I diabetes in a subject, the method comprisingadministering to a subject in need thereof an effective amount of a betapolypeptide composition of the invention. The invention provides for amethod for treating inflammatory bowel disease in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The inventionprovides for a method for treating oophoritis in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of a beta polypeptide composition of the invention. The inventionprovides for a method for treating glomerulonephritis in a subject, themethod comprising administering to a subject in need thereof aneffective amount of a beta polypeptide composition of the invention. Theinvention provides for a method for treating allergic encephalomyelitisin a subject, the method comprising administering to a subject in needthereof an effective amount of a beta polypeptide composition of theinvention. The invention provides for a method for treating myastheniagravis in a subject, the method comprising administering to a subject inneed thereof an effective amount of a beta polypeptide composition ofthe invention.

The invention provides for the use of a population of beta polypeptidesor beta polypeptide molecules, wherein each polypeptide comprises thesequence of SEQ ID NO: 11, 12, 13, 14, 15, or 16, and wherein thepopulation has an average molar ratio of sialic acid groups to betapolypeptide dimer or beta polypeptide molecule of from about 5 to about10 in the manufacture of a medicament for the therapeutic and/orprophylactic treatment of an immune disorder. The invention provides forthe use of a population of beta polypeptides or beta polypeptidemolecules, wherein each polypeptide comprises the sequence of SEQ IDNO11, 12, 13, 14, 15, or 16, and wherein the population has an averagemolar ratio of sialic acid groups to beta polypeptide dimer or betapolypeptide molecule of from about 5 to about 10 in the manufacture ofan anti-rheumatoid arthritis agent in a package together withinstructions for its use in the treatment of rheumatoid arthritis. Inone embodiment, the population has an average molar ratio of sialic acidgroups to beta polypeptide dimer or beta polypeptide molecule of fromabout 5.5 to about 8.5.

Illustrative combination therapies: The invention provides for a methodfor inhibiting T cell proliferation, activation or both, the methodcomprising contacting a T cell with an effective amount of a betapolypeptide composition of the invention in combination withmethotrexate. The invention provides for a method for inhibiting animmune response in a subject, the method comprising administering to asubject in need thereof an effective amount of a beta polypeptidecomposition of the invention in combination with methotrexate. Theinvention provides for a method for inducing immune tolerance to anantigen in a subject, the method comprising administering to a subjectin need thereof an effective amount of a beta polypeptide composition ofthe invention in combination with methotrexate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B presents the nucleotide sequence (SEQ ID NO:1) of a portionof an expression cassette for a CTLA4-Ig molecule. Also shown is theamino acid sequence (SEQ ID NO:2) encoded by the nucleic acid. CTLA4-Igmolecules that can be produced from this expression cassette includemolecules having the amino acid sequence of residues: (i) 26-383 of SEQID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv)26-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQID NO:2. The expression cassette comprises the following regions: (a) anOncostatin M signal sequence (nucleotides 11-88 of SEQ ID NO:1; aminoacids 1-26 of SEQ ID NO:2); (b) an extracellular domain of human CTLA4(nucleotides 89-463 of SEQ ID NO:1; amino acids 27-151 of SEQ ID NO:2);(c) a modified portion of the human IgGl constant region (nucleotides464-1159 of SEQ ID NO:1; amino acids 152-383 of SEQ ID NO:2), includinga modified hinge region (nucleotides 464-508 of SEQ ID NO:1; amino acids152-166 of SEQ ID NO:2), a modified human IgGl C_(H)2 domain(nucleotides 509-838 of SEQ ID NO:1; amino acids 167-276 of SEQ IDNO:2), and a human IgGl C_(H)3 domain (nucleotides 839-1159 of SEQ IDNO:1; amino acids 277-383 of SEQ ID NO:2).

FIG. 2 presents the nucleic acid (top row) and amino acid (bottom row)sequences corresponding to CTLA4^(A29YL104E)-Ig. The amino acid sequencecontains an amino acid change from the sequence shown in FIG. 1, whereinthe changes are at position 29 (A to Y) and at position 10 μL (L to E)compared to that of SEQ ID NO: 2, wherein numbering of amino acidresidues begins at Methionine (M) marked by “+1.” The nucleotidesequence of CTLA4^(A29YL10E)-Ig is shown in this figure starting fromthe A at position 79 (i.e., the position marked by the “+1” below the M)through the A at nucleotide position 1149 (SEQ ID NO:3). In particular,the nucleotide sequence encoding CTLA4^(A29YL104E)-Ig is from thenucleotide at position 79 to the nucleotide at position 1149, designatedSEQ ID NO:3. The full nucleotide sequence shown in FIG. 2 is designatedSEQ ID NO:23 and includes the nucleic acid sequence encoding theOncostatin M signal peptide.

FIG. 3 presents the amino acid sequence (SEQ ID NO:4) ofCTLA4^(A29YL104E)-Ig molecule including an Oncostatin M prosequence (seebold italics). Polypeptides that can be produced that areCTLA4^(A29YL104E)-Ig molecules include molecules having the amino acidsequence of residues: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ IDNO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 26-382 of SEQ ID NO:4, (v)25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4.

FIG. 4 is a model of a CTLA4^(A29YL104E)-Ig shown with the N-linkedglycosylation sites (N76, N108, and N207), the C120-C120 disulfide bond,and the two amino acid substitutions made in the CTLA-4 domain (L104Eand A29Y).

FIG. 5 represents the theoretical cDNA-derived amino acid sequence of aCTLA4^(A29YL104E)-Ig (SEQ ID NO:4). Two amino acid substitutions weremade in the CTLA-4 extracellular domain (L104E and A29Y) to generateCTLA4^(A29YL104E)-Ig. The sequence identifies the signal peptide(pro-sequence) of oncostatin M along with the N-linked glycosylationsites.

FIG. 6 is a graph depicting binding of CTLA4^(A29YL104E)-Ig samples togoat anti-human IgG Fc antibody. Binding of CTLA4^(A29YL104E)-Ig sampleswas detected by measuring the response obtained on this surface,compared to an unmodified sensorchip surface. The various lots representthree different CTLA4^(A29YL104E)-Ig samples.

FIG. 7 is a graph that shows the apparent molecular weights whichcorrespond to multimer, tetramer, and dimer fractions of a CTLA4-Ig HICcleaning peak as determined by an overlay of two-column SEC with dynamiclight scattering detection (DSL) and retention time on SEC.

FIG. 8A (left) and 8B (right) show representative IEF gels of fractionsof glycosylated CTLA4-Ig molecules (comprising SEQ ID NO:2 monomers)isolated and purified from HIC cleaning peak. The loading order for thegel in FIG. 8A is: lane 1, pI markers (Amersham); lane 2, CLTA4-Ig dimerstandard; lane 3, Protein A eluate; lane 4, Multimer; lane 5, tetramer;lane 6, dimer. The loading order for the gel in FIG. 8B is: lane 1, pImarker (Amersham); lane 2, lane 2, CLTA4-Ig dimer standard; lane 3,tetramer; lane 4, dissociated tetramer. The panels show that thetetramer is less sialylated than the dimer.

FIG. 9 shows the predominant carbohydrate structures and relativeamounts of carbohydrates observed on a CTLA4-Ig dimer comprisingmonomers of SEQ ID NO:2. The amino acid residue numbering in the figureis not consistent with SEQ ID NO:2. For the amino acid residue numberingin the figure to be consistent with SEQ ID NO:2, the numeration needs toincrease by 26, i.e., N⁷⁶ is N¹⁰².

FIG. 10 shows a representative IEF gel (pH 4.0 to 6.5) of a CTLA4-Igdimer comprising SEQ ID NO:2 monomers. Lanes 1 and 5 show a calibrationstandard, lane 2, 3, 4 each show 20 μg/μl of CLTA4-Ig dimer.

FIG. 11 shows a representative IEF gel (pH 4.0 to 6.5) of aCTLA4^(A29YL104E)-Ig dimer comprising SEQ ID NO:4 monomers. Lanes 1 and8 show a calibration standard, lane 2-7 each show 10 μg/μl ofCTLA4^(A29YL104E)-Ig dimer.

FIG. 12 shows the N-linked carbohydrate profile of a CTLA4-Ig moleculepopulation comprising monomers of SEQ ID NO:2. The carbohydrates werecollected from glycopeptides and separated using the LC/MS PGC N-linkedOligosaccharide technique. The chromatograms provide the populationprofile for each N-link attachment site. A) The Asn⁷⁶ (Asn¹⁰² of SEQ IDNO:2) carbohydrates from the T5 peptide and B) the Asn¹⁰⁸ (Asn¹³⁴ of SEQID NO:2) carbohydrates from the T7 peptide both show distributions amongmono-and multi-sialylated species. C) The Asn²⁰⁷ (Asn²³³ of SEQ ID NO:2)carbohydrates from the T14 peptide consist of predominantly a sialylatedspecies. D) The distribution of N-linked carbohydrates for CTLA4-Igmolecules is shown. E) A selected raw spectrum from the T5 peptide showsa major peak corresponding to the bi-antennary monosialylated structuredepicted. F) A selected raw spectrum from the T14 shows a major peakcorresponding to the bi-antennary asialo structure. G) A selected rawspectrum consists of a minor species which coelutes with the peak at64.23 minutes, which corresponds to the tri-antennary di-sialylatedstructure. H) A selected raw spectrum reveals the major species in thepeak at 64.23 minutes, which corresponds to the bi-antennarydi-sialylated structure.

FIG. 13A-13B shows a UV and TIC trace of an N-linked oligosaccharideprofile of a CTLA4-Ig SEQ ID NO:2 monomer from PGC chromatography underacidic elution conditions (0.05% TFA). The trace of FIG. 13A showsnegative ion total count (TIC) for PGC chromatograpm under acidicelution conditions (0.05% TFA). The trace of FIG. 13B shows UV trace at206 nm for PGC chromatogram under acidic elutions (0.05% TFA).

FIG. 14A-14B show a UV and TIC trace of an N-linked oligosaccharideprofile of a CTLA4-Ig SEQ ID NO:2 monomer from PGC chromatography underbasic elution conditions (0.4% NH₄OH). The trace of FIG. 14A showsnegative ion total count (TIC) for PGC chromatograpm under basic elutionconditions (0.4% NH₄OH). The trace of FIG. 14B shows UV trace at 206 nmfor PGC chromatogram under basic elutions (0.4% NH₄OH).

FIG. 15 represents the comparative N-linked oligosaccharide carbohydrateprofiles for CTLA4^(A29YL104E)-Ig molecules comprising SEQ ID NO:4. Fouroligosaccharide domains are observed: Domain I contains non-sialylatedspecies, while Domains II, III, and IV contain mono-sialylated,di-sialylated and tri-sialylated species, respectively. Isolating theoligosaccharides chromatographically and analyzing them by massspectroscopy determined the domains.

FIG. 16 shows an HPAEC-PAD profile of N-linked oligosaccharides ofCTLA4-Ig molecules comprising SEQ ID NO:2 monomers. Domains are shown inorder of increasing sialic acid content for oligosaccharides. Domains I,II, III an IV contain oligosaccharide structures having 0, 1, 2, and 3sialic acids respectively. Peak labels represent oligosaccharidestructures assigned by HPAEC-PAD profiling of peaks collected from PGCprofiling. The structural identification of carbohydrate structure isconsistent with previous determinations.

FIG. 17A-17B shows a PGC profile of CTLA4-Ig molecules comprisingmonomers of SEQ ID NO:2. The profile is obtained from direct injectionof carbohydrate digest mixture prepared as described in Example 3.Direct injection results in detection of structure P4144 eluting at 130minutes. The tetra-sialylated structure P4144 is not observed inprofiles of oligosaccharides which are isolated prior to injection.

FIG. 18 presents a LC/MS deconvoluted positive electrospray spectrum forthe T9 fragment of a SEQ ID NO:2 monomer. The spectrum illustrates threemajor O-linked structures. The spectrum illustrates the base peptidewith sugar ladder consistent with the O-linked structure(GalNAc)₁(Gal)₁(NewAc)₁. The bold portion of the spectrum has beenenhanced 10-fold with respect to the non-bold portion of the spectrumand illustrates two additional O-linked structures with(GalNAc)₁(Gal)₁(NeuAc)₂ and (GalNAc)₁(GlcNAc)₁(Gal)₂(NeuAc)₂.

FIG. 19 shows the attachment points and relative populations of O-linkedcarbohydrate structures of a CTLA4-Ig single chain having a SEQ ID NO:2monomer sequence. The relative amounts at each site show data generatedby two or more orthogonal techniques and are subject to variability. Thelocation of the covalent cysteinylation is also depicted.

FIG. 20 depicts a map of the intermediate plasmid piLN-huCTLA4-Ig. Thisplasmid has comprises a sequence that can encode a human CTLA4-Igmolecule (huCTLA4-Ig) (i.e., SEQ ID NO:1) flanked by the restrictionenzyme sites HindIII and XbaI.

FIG. 21 depicts a map of the plasmid pD16 LEA29Y. This plasmid comprisesa sequence that can encode a human CTLA4^(A29YL104E)-Ig molecule (i.e.,SEQ ID NO:4).

FIG. 22 is a photograph of a Southern blot of DNA extracted from CHOcells expressing the CTLA4-Ig expression cassette derived from1D5-100A1(for example, clone 17). The lanes for the gel from left to right are:lane M, DNA molecular weight marker; lane N, EcoRI/XbaI digesteduntransfected CHO DNA (5 mg); lane 1, EcoRI/XbaI digested untransfectedCHO DNA (2.5 μg)+1 ng pcSDhuCTLA4-Ig; lane 2, EcoRI/XbaI digesteduntransfected CHO DNA (2.5 μg)+0.5 ng pcSDhuCTLA4-Ig; lane 3, EcoRI/XbaIdigested untransfected CHO DNA (2.5 μg)+0.25 ng pcSDhuCTLA4-Ig; lane 4,EcoRI/XbaI digested untransfected CHO DNA (2.5 μg)+0.125 ngpcSDhuCTLA4-Ig; lane 5, EcoRI/XbaI digested untransfected CHO DNA (2.5μg)+0.0625 ng pcSDhuCTLA4-Ig; lane 6, EcoRI/XbaI digested untransfectedCHO DNA (2.5 μg)+0.03125 ng pcSDhuCTLA4-Ig; lane 7, EcoRI/XbaI digestedDNA: MCB (5.0 μg); lane 8, EcoRI/XbaI digested DNA: EPCB Lot NumberC20030618A-01 (5.0 μg); lane 9, EcoRI/XbaI digested DNA: EPCB Lot NumberC20030712A-01 (5.0 μg); lane 10, EcoRI/XbaI digested DNA: EPCB LotNumber C20030801A-01 (5.0 μg); lane 11, EcoRI/XbaI digested DNA: MCB(2.5 μg); lane 12, EcoRI/XbaI digested DNA: EPCB Lot NumberC20030618A-01 (2.5 μg); lane 13, EcoRI/XbaI digested DNA: EPCB LotNumber C20030712A-01 (2.5 μg); lane 14, EcoRI/XbaI digested DNA: EPCBLot Number C20030801A-01 (2.5 μg); lane 15, EcoRI/XbaI digested DNA: MCB(1.25 μg); lane 16, EcoRI/XbaI digested DNA: EPCB Lot NumberC20030618A-01 (1.25 μg); lane 17, EcoRI/XbaI digested DNA: EPCB LotNumber C20030712A-01 (1.25 μg); lane18, EcoRI/XbaI digested DNA: EPCBLot Number C20030801A-01 (1.25 μg).

FIG. 23 depicts a flow diagram of a production-scale culturing process.This process allows for the mass-production of recombinant proteins in a25,000-L production bioreactor.

FIG. 24 shows a representative chromatogram of NGNA and NANA systemsuitability standard. The peak at ˜9.7 min is NGNA, and the peak at˜10.7 min is NANA.

FIG. 25 shows a representative chromatogram of hydrolyzed CTLA4-Igmolecules comprising SEQ ID NO:2 monomers. The peak at ˜8.4 min is thesolvent peak. The peak at ˜9.6 min is NGNA. The peak at ˜10.5 min isNANA. The peak at ˜11.3 min is degraded NANA, resulting from thehydrolysis conditions. The area counts of NANA and degraded NANA arecombined for calculations of the NANA molar ratio.

FIGS. 26A, 26B, and 26C show the MALDI spectra of CTLA4-Ig cysteinylatedpeptide. The MALDI spectra were obtained for CTLA4-Igtrypsin/chymotrypsin fragment containing Cys¹⁴⁶ of SEQ ID NO:2. FIG. 26Ashows the single chain peptide spectrum illustrating cysteinylationmodification. FIG. 26B shows the spectrum of the single-chain peptidefollowing reduction and demonstrates that the modification occurs atCys¹⁴⁶. FIG. 26C shows alkylation of the reduced single-chain peptide,which demonstrates that the cysteinylation occurs at Cys¹⁴⁶.

FIG. 27 presents a cloning scheme useful for generating the vector pcSD.pcDNA3 was digested with the restriction enzyme NaeI in order to isolatea 3.821 Kb fragment that contains the CMV promoter, an ampicillinresistance gene, and an origin of replication for E. coli. pSV2-dhfr wasdigested with the restriction enzymes PvuII and BamHI in order toisolate a 1.93 Kb fragment, which contains the SV40 promoter and thedhfr gene, and was subsequently blunt-ended. To generate pcSD, bothfragments were ligated. The map of plasmid pcSD is shown at the bottomof the figure.

FIG. 28 presents a cloning scheme useful for generating the expressionvector pcSDhuCTLA4-Ig. pcSD was digested with the restriction enzymesEcoRV and XbaI. piLN-huCTLA4-Ig was digested with the restriction enzymeHindIII, blunt-ended, and then digested with the restriction enzyme XbaIin order to isolate the 1.2 Kb huCTLA4-Ig fragment. To generatepcSDhuCTLA4-Ig, the CTLA4-Ig fragment was ligated to the digested pcSDvector. The map of plasmid pcSDhuCTLA4-Ig is shown at the bottom of thefigure. This plasmid was linearized and transfected into CHO cells thatdo not have a functional dhfr gene. As the plasmid contains a functionaldhfr gene, stable transfectants can be selected on the basis of cellsurvival. The pcSDhuCTLA4-Ig has the expression cassette comprising theCMV promoter, a sequence that can encode a human CTLA4-Ig molecule(huCTLA4-Ig) (i.e., SEQ ID NO:1) and a poly(A) tail sequence from BGH.

FIG. 29 shows an electropherogram of system suitability aminomonosacchrarides depicted as relative fluorescence units (RFU) versustime (min).

FIG. 30 shows an electropherogram of system suitability neutralmonosacchrarides depicted as relative fluorescence units (RFU) versustime (min).

FIG. 31 represents a tryptic peptide map of CTLA4^(A29YL104E)-Ig withpeptides labeled. Table 23 corresponds with the labeled peptides.

FIG. 32A-32B shows a Northern Hybridization Analysis of theCTLA4^(A29YL104E)-Ig. Panel A depicts an Ethidium bromide-stainedagarose gel wherein Lane M is RNA marker; Lane 1 is total CHO RNA; Lane2 is total MCB RNA; and Lane 3 is total EPCB RNA. Panel B is thecorresponding autoradiogram wherein Lane M is RNA marker; Lane 1 istotal CHO RNA; Lane 2 is total MCB RNA; and Lane 3 is total EPCB RNA.

FIG. 33A-33C depict size exclusion chromatograms, which distinguishCTLA4^(A29YL104E)-Ig dimers from high and low molecular weight species.

FIG. 34 shows an SDS-PAGE (Reduced and Non-Reduced) analysis ofCTLA4^(A29YL104E)-Ig stained with Coomassie Blue. Lane 1 is loaded withmolecular weight markers; Lanes 2, 7, and 12 are blank; Lanes 3-6 areCTLA4^(A29YL104E)-Ig samples (reduced); Lanes 8-11 areCTLA4^(A29YL104E)-Ig samples (non-reduced).

FIG. 35 shows an SDS-PAGE (Reduced and Non-Reduced) analysis ofCTLA4^(A29YL104E)-Ig subjected to silver-staining. Lane 1 is loaded withmolecular weight markers; Lanes 2, 7, and 12 are blank; Lanes 3-6 areCTLA4^(A29YL104E)-Ig samples (reduced); Lanes 8-11 areCTLA4^(A29YL104E)-Ig samples (non-reduced).

FIG. 36 depicts a peptide map of non-reduced CTLA4^(A29YL104E)-Ig usinga combination of trypsin and chymotrypsin digestion.

FIG. 37 depicts a peptide map of non-reduced CTLA4^(A29YL104E)-Ig usinga combination of trypsin and elastase digestion.

FIG. 38 is a diagram that depicts patients with anti-CTLA4-Ig oranti-CTLA-4 responses. Antibody response to the whole CTLA4-Ig molecule(CTLA-4 and Ig portion) and the CTLA-4 portion only were determinedusing Assays A and B, as outlined in the Example 32.

FIG. 39 is a schematic demonstrating the distribution of clearance andthe volume of central compartment by immunogenicity status.

FIG. 40 is a graph demonstrating profiles of mean (SD) CTLA4-Ig serumconcentrations over time in monkeys adminstered 10 mg/kg of drugsubstance produced by a process of the invention.

FIG. 41 is a graph of a Size Exclusion Chromatography (SEC) chromatogramof Protein A (MAbSelect) purified from control and disaggregatedCTLA4-Ig material.

FIG. 42 is a graph of an N-glycan analysis comparing the DisaggregationProcessed Material (ii) to Control (i).

FIG. 43 is a graph depicting the mean CTLA4-Ig serum concentrations[μg/ml] versus time (over 71 days).

FIG. 44 shows an electropherogram of neutral monosacchrarides depictedas relative fluorescence units (RFU) versus time (min).

FIG. 45 shows an electropherogram of amino monosacchrarides depicted asrelative fluorescence units (RFU) versus time (min).

FIG. 46 represents the comparative N-linked oligosaccharide carbohydrateprofiles for CTLA4^(A29YL104E)-Ig molecules comprising SEQ ID NO:4. Fouroligosaccharide domains are observed, wherein Domain I containsnon-sialylated species, while Domains II, III, and IV containmono-sialylated, di-sialylated and tri-sialylated species, respectively.

FIG. 47 is a graph of a capillary electrophoretic separation of CTLA4-Igthat was mixed 1:1 with CTLA4^(A29YL104E)-Ig. The main peak migrationtimes are approximately 0.8 minutes apart.

FIG. 48 is a chromatogram of hydrolyzed CTLA4^(A29YL104E)-Ig material,wherein a NANA Peak is observed at 3.4 minutes.

FIG. 49 depicts several of the various N-linked carbohydrate structuresfound in mammalian proteins. All chains share a common core structurecontaining two GlcNAc and three mannose residues.

FIG. 50 is graph depicting CTLA4-Ig exposure (AUC) as a function ofsialylation of the glycoprotein (NANA ratio).

FIG. 51 is graph depicting CTLA4-Ig exposure (AUC) as a functionCTLA4-Ig's carbohydrate profile. A large number of peaks were generatedby anion exchange HPLC which were resolved into four or five domains.Domains 1 and 2 are largely asialylated and mono-sialylated structures,while domains 3 and 4 are largely di-and tri-sialylated structures.

FIG. 52 represents a tryptic peptide map of CTLA4^(A29YL104E)-Ig withpeptides labeled. Table 56 corresponds with the labeled peptides.

FIG. 53 represents the comparative N-linked oligosaccharide carbohydrateprofiles for CTLA4-Ig molecules comprising SEQ ID NO:2. Fouroligosaccharide domains are observed, wherein Domain I containsnon-sialylated species, while Domains II, III, and IV containmono-sialylated, di-sialylated and tri-sialylated species, respectively.

FIG. 54A-54D is a graph that represents the oligosaccharide profiles ofCTLA4-Ig and Peptides T5, T7, and T14 by HPAEC-PAD.

FIG. 55 is a graph depicting the labeled oligosaccharide profile ofCTLA4-Ig obtained from PGC (Hypercarb) Column.

FIG. 56 depicts a graph of pharmacokinetic data showing monkey AUC onthe Y axis and percent of N-linked glycosylation as shown in Domains Iand II from a carbohydrate profile on the X axis. See methods ofdetermining the N-linked carbohydrate profile in, for example, Examples3, 44, 22 and 37. As the percentage of Domains I and II increases (andthe percentage of Domains III, IV and V decreases), clearance increases.Note that the negative control, the CTLA4-Ig with low sialic acid iscleared very rapidly. Note that the CTLA4-Ig variant, LEA(CTLA4-Ig^(A29YL104E)-Ig) is included in this graph.

FIG. 57A and 57B depict a trace of a N-linked carbohydrate chromatogramof the N-linked carbohydrates released from CTA4-Ig (as obtained frommethods such as those described in Examples 3, 44, 22 and 37). The tracein FIG. 57A is of from an analysis of CTLA4-Ig produced in a culturemethod without additional galactose added to the culture. The trace inFIG. 57B did have galactose added to the culture. The percentages ofN-linked carbohydrates in each Domain is shown in the inset table.

FIG. 58 depicts a trace of a N-linked carbohydrate chromatogram of theN-linked carbohydrates released from CTA4-Ig (as obtained from methodssuch as those described in Examples 3, 44, 22 and 37). This is from ananalysis of CTLA4-Ig produced in a culture method with galactose addedto the culture at day 8. This trace of from an analysis of CTLA4-Igproduced in a culture method without additional galactose added to theculture. The percentages of N-linked carbohydrates in each Domain isshown in the inset table.

FIG. 59 depicts a trace of a N-linked carbohydrate chromatogram of theN-linked carbohydrates released from CTA4-Ig (as obtained from methodssuch as those described in Examples 3, 44, 22 and 37). This is from ananalysis of CTLA4-Ig produced in a culture method with galactose addedto the culture at day 14. This trace of from an analysis of CTLA4-Igproduced in a culture method without additional galactose added to theculture. The percentages of N-linked carbohydrates in each Domain isshown in the inset table.

FIG. 60A and 60B depicts a trace of a N-linked carbohydrate chromatogramof the N-linked carbohydrates released from CTA4-Ig (as obtained frommethods such as those described in Examples 3, 44, 22 and 37). FIG. 60Ais from an analysis of CTLA4-Ig produced in a culture method withoutgalactose added, and FIG. 60B is from an analysis where galactose wasadded to the culture at day 14. This trace of from an analysis ofCTLA4-Ig produced in a culture method without additional galactose addedto the culture. The percentages of N-linked carbohydrates in each Domainis shown in the inset table.

FIG. 61 depicts a trace of a N-linked carbohydrate chromatogram of theN-linked carbohydrates released from CTA4-Ig (as obtained from methodssuch as those described in Examples 3, 44, 22 and 37). This trace wasobtained from CTLA4-Ig material that was recovered from the wash step ofthe QFF column, producing a cut of CTLA4-Ig material with low sialicacid. The relative amount of Domain I and II is increased and DomainsIII and Iv are decreased, compared to the traces shown in FIGS. 60, 59and 58. The percentages of N-linked carbohydrates in each Domain isshown in the inset table.

FIG. 62 shows the tryptic peptide map of CTLA4-Ig indicating that T8elutes at the end of the solvent front, and T9 elutes at the shoulder ofT27.

FIG. 63 is a graph that represents the full mass spectrum correspondingto glycopeptide T8 from CTLA4-Ig.

FIG. 64 is a graph that represents the full mass spectrum correspondingto glycopeptide T9 from CTLA4-Ig.

FIG. 65 is a graph that represents the MALDI-TOF data for the T9 peptidefragment from CTLA4-Ig.

FIG. 66A-66B depicts ion chromatograms and mass spectra of oxidized andnative tryptic peptides from CTLA4-Ig.

FIG. 67 depicts a typical N-Linked Oligosaccharide Profile (Domains I,II, III, IV and V, and Peaks 1A and 1B within 5% of Lot averages). Peaks1A, 1B and 1C represent the asialo N-linked oligosaccharide structuresof G0, G1 and G2. The data for the profile is in the table directlybelow. See Example 44.

Peak Name RT Area % Area 1 Domain I 19.413 47807873 31.3 2 Domain II29.076 50746179 33.2 3 Domain III 42.819 36640805 24.0 4 Domain V 67.5463421324 2.2 5 Domain IV 55.899 14331509 9.4 6 Peak 1A 19.413 111151687.3 7 Peak 1B 20.290 16331761 10.7 8 Peak 1C 21.032 13507144 8.8 9 Peak2 21.925 4285962 2.8 10 22.685 2567838 1.7 11 29.076 2808537 1.8 1230.763 27989176 18.3 13 31.577 19948466 13.0 14 42.819 4555254 3.0 15Peak 3 43.823 22213064 14.5 16 46.626 9872487 6.5 17 55.899 3898179 2.518 Peak 4 57.368 6789516 4.4 19 60.333 3643813 2.4 20 67.546 3421324 2.2

FIG. 68 depicts an isoelectric focusing gel of CTLA4-Ig. The bands arecharacterized by:

Relative Cumulative Band Protein Load Band % Band Percent LaneDescription (micrograms) No. Intensity (%) 1 IEF Markers NA NA NA NA 2CTLA4-Ig 20 16 100 NA material 3 CTLA4-Ig Drug 20 16 100 100 Substance 4Staining 1 NA NA NA Control

FIG. 69 depicts a representative isoelectric focusing gel quantitativeanalysis report of CTLA4-Ig. The quantitation of the gel was performedand the data is as follows:

BQC060082 IEF Marker Lot 188667-2003-015 (DS ABC04014) Staining ControlLane 1 Lane 1 Lane 1 Lane 2 Lane 2 Lane 2 Lane 3 Lane 3 Lane 3 Lane 4Lane 4 Lane 4 Band Band % pI Band Band % pI Band Band % pI Band Band %pI 1 20.95 5.85 1 0.51 5.28 1 1.34 5.26 1 100 5.68 2 22.39 5.2 2 0.175.25 2 2.34 5.19 3 9.62 4.55 3 0.71 5.23 3 0.99 5.17 4 47.04 3.5 4 1.615.2 4 3.2 5.14 5 1.15 5.18 5 3.23 5.11 6 7.44 5.15 6 3.41 5.08 7 10.965.09 7 10.29 5.03 8 9.32 5.02 8 5.95 5.01 9 18.22 4.94 9 5.66 4.97 105.52 4.91 10 13.4 4.91 11 4.19 4.88 11 17.11 4.85 12 22.58 4.82 12 7.394.8 13 3.8 4.67 13 16.03 4.72 14 10.02 4.63 14 3.88 4.56 15 3.44 4.55 154.97 4.52 16 0.35 4.48 16 0.8 4.45 Bands (4.3-5.6) 16 Bands (4.3-5.6) 16% Bands (4.3-5.3) 100 % Bands (4.3-5.3) 100 Sample Relative Percent (%)100 Sample Relative Percent (%) = (Sample % Band Intensity/Ref % BandIntensity) × 100 NOTE: For the pI range of 4.3 to 5.3

FIGS. 70A-70B. FIG. 70A depicts Typical 20 μL Injection of SystemSuitability Standard on TOSO HAAS 3000 SWXL Column Equipped with a GuardColumn. FIG. 70B depicts a 20 μL Injection of CTLA4-Ig ReferenceMaterial on TOSO HAAS 3000 SWXL Column Equipped with a Guard Column.

FIG. 71—Example of digitally acquired image of SDS-PAGE Analysis ofCTLA4-Ig by Coomassie Blue stained Polyacrylamide (4-20) GelElectrophoresis

Non-Reduced Percent Protein Load (NR)/Reduced (%) Band Lane Description(micrograms) (R) Condition Intensity 1 CTLA4-Ig drug 10 NR 100 substance2 CTLA4-Ig material 10 NR  99 3 Blank NA NR NA 4 CTLA4-Ig drug 10 R 100substance 5 CTLA4-Ig material 10 R 100 6 Molecular Weight NA NA NAMarker 7 CTLA4-Ig drug 10 NR  99 product 8 CTLA4-Ig material 10 NR 100 9Blank NA NR NA 10 CTLA4-Ig drug 10 R  99 product 11 CTLA4-Ig material 10R  99 12 Trypsin Inhibitor NA NA NA Staining Control

FIG. 72 depicts an example of quantitative analysis report for CoomassieBlue stained SDS-PAGE.

FIG. 73 shows a table setting out the quantitative analysis of thestained SDS-PAGE gel in FIG. 72.

FIG. 74 depicts example of an enhanced image of SDS-PAGE Analysis ofCTLA4-Ig Coomassie Blue Stained Gel for illustrating the migratingpositions of the major and expected minor bands relative to theMolecular Weight Markers.

FIG. 75 is a depiction of a representative N-Linked Carbohydrate Profileof CTLA4-Ig reference/standard material. This is a representativecarbohydrate profile of run on the Waters system. Retention times aresystem dependent.

FIG. 76 is a depiction of a representative Stachyose System SuitabilityChromatogram.

FIG. 77 is a trace of a Representative Chromatogram of HydrolyzedCTLA4-Ig Material.

FIG. 78 depicts a ccanned gel image of SDS-PAGE Analysis ofCTLA4^(A29YL104E)-Ig Coomassie Blue Stained Polyacrylamide (4-20%) GelElectrophoresis Coomassie Blue Staining.

Protein Load Condition % Purity Lane# Description (μg) R/NR (%) 1CTLA4^(A29YL104E)-Ig Drug 10 NR 99 Substance 2 CTLA4^(A29YL104E)-Ig 10NR 99 Reference Material 3 Blank NA NR NA 4 CTLA4^(A29YL104E)-Ig Drug 10R 100 Substance 5 CTLA4^(A29YL104E)-Ig 10 R 100 Reference Material 6Molecular Weight Marker NA R NA 7 CTLA4^(A29YL104E)-Ig 10 R 99 ReferenceMaterial 8 CTLA4^(A29YL104E)-Ig Drug 10 R 99 Product 9 Blank 0 NR 0 10CTLA4^(A29YL104E)-Ig 10 NR 100 Reference Material 11CTLA4^(A29YL104E)-Ig Drug 10 NR 100 Product 12 Staining control 0.1 NR100

FIG. 79 depicts a flow diagram of the harvest steps, see Example 28.

FIG. 80 depicts an electropherogram of system suitability of aminomonosaccharides. See Example 16.

FIG. 81 depicts a graph of pharmacokinetic data showing monkey AUC onthe Y axis and percent of N-linked glycosylation as shown in Domains Iand II from a carbohydrate profile on the X axis. See methods ofdetermining the N-linked carbohydrate profile in, for example, Examples3, 44, 22 and 37. As the percentage of Domains I and II increases (andthe percentage of Domains III, IV and V decreases), AUC increases. Notethat the negative control, the CTLA4-Ig with low sialic acid is clearedvery rapidly. Note that the mutant CTLA4-Ig molecules,CTLA4-Ig^(A29YL104E)-Ig (designated LEA) is included in this graph.

FIG. 82 depicts a graph of pharmacokinetic data showing AUC on the Yaxis and percent of N-linked glycosylation as shown in Domains III andIV (as determined from an N-linked carbohydrate profile) on the X-axis.As the percentage of Domains III and IV increase, the AUC increases.Note that the negative control, the CTLA4-Ig with low sialic acid iscleared very rapidly. See methods of determining the N-linkedcarbohydrate profile in, for example, Examples 3, 44, 22 and 37. Notethat the mutant CTLA4-Ig molecules, CTLA4-Ig^(A29YL104E)-Ig (designatedLEA) is included in this graph.

FIG. 83 depicts another graph of pharmacokinetic data showing AUC on theY axis and percent of N-linked glycosylation as shown in Domains III andIV from a carbohydrate profile on the X-axis. As the percentage ofDomains III and IV increase, the AUC increases. Note that the negativecontrol, the CTLA4-Ig with low sialic acid is cleared very rapidly. Seemethods of determining the N-linked carbohydrate profile in, forexample, Examples 3, 44, 22 and 37. Note that the mutant CTLA4-Igmolecules, CTLA4-Ig^(A29YL104E)-Ig (designated LEA) is included in thisgraph.

FIG. 84 depicts a tryptic Map of CTLA4-Ig standard see Table at end ofExample 65 for peak assignments. The small peak labeled T1+A is the T1tryptic peptide extended by an N-terminal alanine residue. The smallpeak labeled T31+K is the T31 tryptic peptide extended by a C-terminallysine residue.

FIG. 85 depicts an overlay of 280 nm Data for Tryptic Map of CTLA4-IgStandard Plus Same Spiked with 5 mole % of T6ox Indicator Peptide,Met(O) 85 (84-93). See Example 65.

FIG. 86 depicts an expanded View of 215 nm Data for Tryptic Map ofCTLA4-Ig Standard Plus Same Spiked with 5 mole% of T26deam1 IndicatorPeptide, isoAsp294(281-302). See Example 65.

FIG. 87 provides the legend for naming the oligosaccharides in FIG. 88.The grey shaded area shows the N-linked oligosaccharide core structure,where P stands for PNGase F digestion, and the subsequent digitsdescribe the number of Mannose (circle), Fucose (downwards pointingtriangle), Galactose (right pointing triangle), and sialic acid residues(star), respectively. N-acetyl glucosamine (GlcNAc) is represented by asquare.

FIG. 88A-88C shows the N-linked oligosaccharide structures and massesdetected in CTLA4-Ig.

FIG. 89 is a flow diagram showing an example of a purification processof CTLA4^(A29YL104E).

FIG. 90 is a flow diagram showing an overview of the procedure forcarbohydrate content analysis of a CTLA^(A29YL104E)-Ig composition,tryptic peptide mapping and IEF.

FIG. 91 is a flow diagram for the CTLA4-Ig production process.

FIG. 92 is a flow diagram for the downstream steps of CTLA4-Igproduction process.

FIG. 93 is a flow diagram of the procedure for an in-vitro cell basedbioassay for CTLA4^(A29YL104E)-Ig.

FIG. 94 is an outline for a method for determination of bio-specificbinding of CTLA4^(A29YL104E)-Ig to the B7.1-Ig receptor by surfaceplasmon resonance (BIAcore).

FIG. 95 is a flow diagram of the procedure for an in-vitro cell basedbioassay for CTLA4^(A29YL104E)-Ig.

FIG. 96 shows oligosaccharide structures of Peptide T8.

FIG. 97 shows oligosaccharide structures of Peptide T9.

FIG. 98 is a method outline for determination of Chinese hamster ovary(CHO) host cell protein impurities in CTLA4^(A29YL104E)-Ig drugsubstance by ELISA.

FIG. 99 is a method outline for determination of Protein A levels inCTLA4^(A29YL104E)-Ig by ELISA.

DETAILED DESCRIPTION OF THE INVENTION

CTLA4-Ig molecules can be used to treat a variety of disorders,including disorders relating to aberrant immunoproliferative andimmunoreactive phemonena such as autoimmunity and allergy. The inventionprovides CTLA4-Ig compositions that comprise, for example, populationsof CTLA4-Ig molecules having particular glycosylation modifications,having particular carbohydrate profiles or characteristics, havingparticular multimeric structures, and/or having particular aviditystrengths. Documents that are hereby incorporated by reference in theirentirety that also describe CTLA4-Ig molecules, uses and methodsthereof, include U.S. Pat. Nos. 5,434,131; 5,851,795; 5,885,796;5,885,579; and 7,094,874.

The invention also provides cell lines that are capable of producinglarge amounts of CTLA4-Ig molecules via the mass-production andculturing methods provided herein. One particular cell line of theinvention is a clonal cell line that can be used to mass-produceCTLA4-Ig molecules such that it has a particular glycosylation andcarbohydrate profile. As compared to the heterogeneous and non-clonalcell population having ATCC Accession No. 68629 (see U.S. Pat. No.5,434,131, which is hereby incorporated by reference in its entirety),the clonal cell lines of the invention can secrete a population ofCTLA4-Ig molecules having a more consistent or more uniformglycosylation or carbohydrate profile. Further, as compared to theheterogeneous and non-clonal cell population having ATCC Accession No.68629, the clonal cell lines of the invention can secrete a greateramount of CTLA4-Ig molecules, in part because the present clonal celllines are selected to have a high-copy number of CTLA4-Ig expressioncassettes integrated into a single site in the genome of the cell.

The invention provides for the discovery that the avidity and potency ofCTLA4-Ig (SEQ ID NO:2) can be increased by making two amino acidsubstitutions in the B7 binding region of the CTLA-4 binding domain: (i)alanine at position 29 of SEQ ID NO:2 is substituted by tyrosine (A29Y),and (ii) lysine at position 10 μL of SEQ ID NO:2 is substituted byglutamate (L104E). The invention provides a subgenus of CTLA4-Igmolecules, called “beta polypeptide molecules,” which comprise betapolypeptides which have B7 binding activity and may comprise the aminoacid sequence in SEQ ID NO: 24 (CTLA4 extracellular domain with A29Y andL104E mutations), linked to an immunoglobulin constant region, orportion thereof.

[SEQ ID NO: 24] MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSD A CTLA4 extracellular domain [SEQ ID NO: 18]MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD

Terms

As used herein, the term “clonal” refers to a cell population that isexpanded from a single cell. With respect to a clonal cell line orclonal cell population capable of expressing a CTLA4-Ig molecule, theclonal cell line or population is expanded from a single cell that wasisolated from a population of cells that were transfected with anexpression vector encoding the CTLA4-Ig molecule. The transfectedpopulation of cells can be a heterogeneous population. A clonal cellline or population can be considered to be homogeneous in the sense thatall of the cells in the population came from a single transfectant.

As used herein, the term “B7-1” refers to CD80; the term “B7-2” refersCD86; and the term “B7” refers to either or both of B7-1 and B7-2 (CD80and CD86). The term “B7-1-Ig” or “B7-1Ig” refers to CD80-Ig; the term“B7-2-Ig”or “B7-2Ig” refers CD86-Ig.

As used herein, the terms “CTLA4-Ig” or “CTLA4-Ig molecule” or “CTLA4Igmolecule” or “CTLA4-Ig protein” or “CTLA4Ig protein” are usedinterchangeably, and refer to a protein molecule that comprises at leasta CTLA4-Ig polypeptide having a CTLA4 extracellular domain and animmunoglobulin constant region or portion thereof. In some embodiments,for example, a CTLA4-Ig polypeptide comprises at least the amino acidsequence of SEQ ID NO:18. In certain embodiments, the CTLA4extracellular domain and the immunoglobulin constant region or portionthereof can be wild-type, or mutant or modified. A mutant CTLA4-Igpolypeptide is a CTLA4-Ig polypeptide comprising a mutant CTLA4extracellular domain. A mutant CTLA4Ig molecule comprises at least amutant CTLA4-Ig polypeptide. In some embodiments, the CTLA4extracellular domain and the immunoglobulin constant region or portionthereof can be mammalian, including human or mouse. In some embodiments,a mutant CTLA4 extracellular domain can have an amino acid sequence thatis at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical tothe CTLA4 extracellular domain shown in FIG. 1 or SEQ ID NO:18. In someembodiments, a mutant immunoglobulin constant region or portion thereofcan have an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical to the immunoglobulin (g) constantregion as shown in FIG. 1. The polypeptide can further compriseadditional protein domains. A CTLA4-Ig molecule can refer to a monomerof the CTLA4-Ig polypeptide, and also can refer to multimer forms of thepolypeptide, such as dimers, tetramers, and hexamers, etc. (or otherhigh molecular weight species). CTLA4-Ig molecules are also capable ofbinding to CD80 and/or CD86. CTLA4-Ig molecules include mutant CTLA4Igmolecules, such as “beta polypeptides molecules,” e.g.,CTLA4^(A29YL104E)-Ig. For example, CTLA4-Ig comprises CTLA4-Igmolecules, and CTLA4^(A29YL104E)-Ig comprises beta polypeptidesmolecules (an example of mutant CTLA4-Ig molecules).

As used herein, the term “CTLA4 extracellular domain” refers to aprotein domain comprising all or a portion of the amino acid sequenceshown in SEQ ID NO:18, that binds to B7-1 (CD80) and/or B7-2 (CD86). Insome embodiments, a CTLA4 extracellular domain can comprise apolypeptide having an amino acid sequence that is at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 27-150 ofSEQ ID NO:2, which are the same as amino acids shown SEQ ID NO:18. Theamino acid 151 of SEQ ID NO:2 is a junction amino acid.

As used herein, the term “beta polypeptide” refers to a mutant CTLA4-Igpolypeptide that (1) comprises the amino acid sequence of SEQ ID NO:18wherein the amino acid at position 29 is mutated to tyrosine and theamino acid at position 10 μL is mutated to glutamate, optionally withvarious additional mutations, and an immunoglobulin constant region, ora portion thereof; and (2) is capable of binding to CD80 and/or CD86. Insome embodiments, for example, a beta polypeptide comprises at least theamino acid sequence of the extracellular domain of CTLA4^(A29YL104E)-Ig(as shown in SEQ ID NO:24). Non-limiting examples of beta polypeptidesinclude belatacept and SEQ ID NOS: 4 and 11-16. In certain embodiments,the immunoglobulin constant region or portion thereof can be wild-type,or mutant or modified. In certain embodiments, the immunoglobulinconstant region or portion thereof can be mammalian, including human ormouse. Additional non-limiting examples of beta polypeptides include abeta polypeptide comprising one or more amino acid mutations in theimmunoglobulin constant region or portion thereof (for example,substitution of cysteine 120 of SEQ ID NO:4), and a beta polypeptidecomprising further mutations at one or more of amino acid position 25,30, 93, 96, 103 or 105 of SEQ ID NO:18. A beta polypeptide moleculecomprises a beta polypeptide. A beta polypeptide molecule can refer to amonomer of the beta polypeptide and smultimer forms of the betapolypeptide, such as dimers, tetramers and hexamers, etc. For example,belatacept comprises beta polypeptide molecules. Beta polypeptidemolecules are further described in U.S. Provisional Application No.60/849,543 filed on Oct. 5, 2006, which is hereby incorporated byreference in its entirety.

As used herein, the terms “glutamate” and “glutamic acid” are usedinterchangeably.

As used herein, the term “dimer” refers to a CTLA4-Ig protein orCTLA4-Ig molecule composed of two CTLA4-Ig polypeptides or monomerslinked or joined together. The linkage between monomers of a dimer canbe a non-covalent linkage or interaction, a covalent linkage orinteraction, or both. An example of a CTLA4-Ig dimer is shown in FIG. 4.A CTLA4-Ig protein or CTLA4-Ig molecule composed of two identicalmonomers is a homodimer. A CTLA4-Ig homodimer also encompasses amolecule comprising two monomers that may differ slightly in sequence. Ahomodimer encompasses a dimer where the monomers joined together havesubstantially the same sequence. The monomers comprising a homodimershare considerable structural homology. For example, the differences insequence may be due to N-termal processing modifications of the monomer.

As used herein, “conservative mutation” refers to a change in a nucleicacid sequence that substitutes one amino acid for another of the sameclass (e.g., substitution of one nonpolar amino acid for another, suchas isoleucine, valine, leucine, or methionine; or substitution of onepolar amino acid for another, such as substitution of arginine forlysine, glutamic acid for aspartic acid or glutamine for asparagine).

As used herein, “non-conservative mutation” refers to a change in anucleic acid sequence that substitutes one amino acid for another of adifferent class (e.g., substitution of one basic amino acid, such aslysine, arginine or histidine, with an acidic amino acid, such asaspartic acid or glutamic acid). For example, an amino acid can bebiochemically dissimilar from another amino acid based on size, charge,polarity, reactivity or other such characteristics of amino acids.

As used herein, “isolated” refers to a molecule that is taken out of itsnative environment and is in an environment different from that in whichthe molecule naturally occurs, or a substance (e.g., a protein) that ispartially or completely recovered or separated from other components ofits environment such that the substance (e.g., protein) is thepredominant species (e.g., protein species) present in the resultantcomposition, mixture, or collection of components (for example, on amolar basis it is more abundant than any other individual species in thecomposition). For example, a preparation may consist of more than about70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94 or or 95%, of isolated CTLA4-Ig. “Isolated”does not exclude mixtures of CTLA4-Ig molecules with other CTLA4-Igmolecules from the environment in which the molecule naturally occurs.“Isolated” does not exclude pharmaceutically acceptable excipientscombined with CTLA4-Ig, wherein the CTLA4-Ig has been recovered from itsenvironment, such as a cell culture, a batch culture, or a bioreactor,etc. As used herein, “isolating” refers to carrying out a process ormethod to obtain an isolated CTLA4-Ig molecule.

As used herein, the term “soluble CTLA4” means a molecule that cancirculate in vivo or CTLA4 which is not bound to a cell membrane. Forexample, the soluble CTLA4 can include CTLA4-Ig which includes theextracellular region of CTLA4, linked to an Ig.

As used herein, the term “soluble fraction of a cell culture” refers tothe liquid portion of a cell culture other than, or which issubstantially free of, insoluble, particulate or solid components of thecell culture, such as cells, cell membranes and nuclei. The solublefraction may be, for example, the resulting supernatant followingcentrifugation of the cell culture, or the resulting filtrate followingfiltration of the cell culture.

As used herein, the term “expression cassette” refers to a nucleic acidhaving at least a 5′ regulatory region (e.g., promoter) operably linkedto a nucleotide sequence that encodes a polypeptide, and optionally anuntranslated 3′ termination region (e.g., stop codon and polyadenylationsequence). Under appropriate conditions, a polypeptide encoded by anexpression cassette is produced by the expression cassette. Anexpression cassette may also have one or more nucleotide sequences thattarget integration of the expression cassette into a specific site inthe genome of a host cell (for example, see Koduri et al., (2001) Gene280:87-95). For example, a CTLA4^(A29YL104E)-Ig polypeptide expressioncassette derived from a plasmid deposited as ATCC Accession No.PTA-2104, is one example of an expression cassette encoding aCTLA4^(A29YL104E)-Ig.

As used herein, the term “substantially purified” refers to acomposition comprising a CTLA4-Ig molecule or a selected population ofCTLA4-Ig molecules that is removed from its natural environment (e.g.,is isolated) and is at least 90% free, 91% free, 92% free, 93% free, 94%free, 95% free, 96% free, 97% free, 98% free, 99% free, 99.5% free, or99.9% free from other components, such as cellular material or culturemedium, with which it is naturally associated. For example, with respectto a recombinantly produced CTLA4-Ig protein molecule, the term“substantially purified” can also refer to a composition comprising aCTLA4-Ig protein molecule that is removed from the productionenvironment such that the protein molecule is at least 90% free, 91%free, 92% free, 93% free, 94% free, 95% free, 96% free, 97% free, 98%free, 99% free, 99.5% free, or 99.9% free from protein molecules whichare not polypeptides of SEQ ID NO: 2 or mutant polypeptides of SEQ IDNO: 2 which are of interest. “Substantially purified” does not excludemixtures of CTLA4-Ig molecules (such as dimers) with other CTLA4-Igmolecules (such as tetramer). “Substantially purified” does not excludepharmaceutically acceptable excipients or carriers combined withCTLA4-Ig molecules, wherein the CTLA4-Ig molecules have been taken outof their native environment.

As used herein, the term “large-scale process” is used interchangeablywith the term “industrial-scale process”. The term “culture vessel” isused interchangeably with “bioreactor”, “reactor” and “tank”.

A “liquid culture” refers to cells (for example, bacteria, plant,insect, yeast, or animal cells) grown on supports, or growing suspendedin a liquid nutrient medium.

A “seed culture” refers to a cell culture grown in order to be used toinoculate larger volumes of culture medium. The seed culture can be usedto inoculate larger volumes of media in order to expand the number ofcells growing in the culture (for example, cells grown in suspension).

As used herein, “culturing” refers to growing one or more cells in vitrounder defined or controlled conditions. Examples of culturing conditionswhich can be defined include temperature, gas mixture, time, and mediumformulation

As used herein, “expanding” refers to culturing one or more cells invitro for the purpose of obtaining a larger number of cells in theculture.

As used herein, “population” refers to a group of two or more molecules(“population of molecules”) or cells (“population of cells”) that arecharacterized by the presence or absence of one or more measurable ordetectable properties. In a homogeneous population, the molecules orcells in the population are characterized by the same or substantiallythe same properties (for example, the cells of a clonal cell line). In aheterogeneous population, the molecules or cells in the population arecharacterized by at least one property that is the same or substantiallythe same, where the cells or molecules may also exhibit properties thatare not the same (for example, a population of CTLA4-Ig molecules havinga substantially similar average sialic content, but having non-similarmannose content).

As used herein, “high molecular weight aggregate” is usedinterchangeably with “high molecular weight species” to refer to aCTLA4-Ig molecule comprising at least three CTLA4-Ig monomers. Forexample, a high molecular weight aggregate may be a tetramer, a pentameror a hexamer.

“Percent (%) yield” refers to the actual yield divided by thetheoretical yield, and that value multipled by 100. The actual yield canbe given as the weight in gram or in mol (for example, a molar yield).The theoretical yield can be given as the ideal or mathematicallycalculated yield.

As used herein, an “amount of MCP-1” refers to (1) an amount of MCP-1(Monocyte chemotactic protein-1, especially, hamster MCP-1) alone, or(2) an amount of “MCP-1 like” protein, wherein “MCP-1 like” proteinincludes MCP-1, together with proteins homologous to MCP-1, fragments ofMCP-1, and/or fragments of proteins homologous to MCP-1 (for example, ineach of the aforementioned instances, as may be cross-reactive with anantibody (e.g., polyclonal ELISA) assay for the detection of MCP-1). Theabsence of MCP-1 (and/or proteins homologous to MCP-1, fragments ofMCP-1, and/or fragments of proteins homologous to MCP-1) is contemplatedwhere no lower limit is provided with regard to a range of amounts ofMCP-1.

As used herein, “glycosylation content” refers to an amount of N-linkedor O-linked sugar residues covalently attached to a protein molecule,such as a glycoprotein like a CTLA4-Ig molecule.

As used herein, the term “molar ratio of sialic acids to protein” iscalculated and given as number of moles of sialic acid molecules permoles of protein (CTLA4-Ig molecules) or dimer.

As used herein, the term “glycoprotein” refers to a protein that ismodified by the addition of one or more carbohydrates, including theaddition of one or more sugar residues.

As used herein, the term “sialylation” refers to the addition of asialic acid residue to a protein, including a glycoprotein.

As used herein, the term “glycoprotein isoform” refers to a moleculecharacterized by its carbohydrate and sialic acid content as determinedby isoelectric focusing (IEF) gel electrophoresis or other suitablemethods for distinguishing different proteins in a mixture by theirmolecular weight, charge, and/or other characteristics. For example,each distinct band observed on an IEF gel represents molecules that havea particular isoelectric point (pI) and thus the same net overallcharge. A glycoprotein isoform can be a distinct band observed on an IEFgel where each band can be a population of molecules that have aparticular pI.

“Immune tolerance” refers to a state of unresponsiveness to a specificantigen or group of antigens to which a person is normally responsive(for example, a state in which a T cell can no longer respond toantigen).

“Potency” refers to a measure of the response as a function of ligandconcentration. For example, agonist potency is quantified as theconcentration of ligand that produces half the maximal effect (EC₅₀). Anon-limiting pharmacological definition of potency includes componentsof affinity and efficacy, where, efficacy is the ability of a drug toevoke a response once bound. Potency is related to affinity, but potencyand affinity are different measures of drug action.

As used herein, “pharmaceutically acceptable carrier” refers to avehicle for a pharmacologically active agent. The carrier facilitatesdelivery of the active agent to the target site without terminating thefunction of the agent. Non-limiting examples of suitable forms of thecarrier include solutions, creams, gels, gel emulsions, jellies, pastes,lotions, salves, sprays, ointments, powders, solid admixtures, aerosols,emulsions (e.g., water in oil or oil in water), gel aqueous solutions,aqueous solutions, suspensions, liniments, tinctures, and patchessuitable for topical administration.

As used herein, the phrase “pharmaceutically acceptable composition” (or“pharmaceutical composition”) refers to a composition that is acceptablefor pharmaceutical administration, such as to a human being. Such acomposition can include substances that are impurities at a level notexceeding an acceptable level for pharmaceutical administration (suchlevel including an absence of such impurities), and can includepharmaceutically acceptable excipients, vehicles, carriers and otherinactive ingredients, for example, to formulate such composition forease of administration, in addition to any active agent(s). For example,a pharmaceutically acceptable CTLA4-Ig composition can include MCP-1 orDNA, so long as those substances are at a level acceptable foradministration to humans.

“Drug substance” is the active pharmaceutical ingredient contained in apharmaceutical composition. The term “drug substance” includes an activepharmaceutical ingredient in solution and/or in buffered form. “Drugproduct” is a pharmaceutical composition containing drug substanceformulated for pharmaceutical administration. For purposes of the assayscontained in the Examples and elsewhere herein, which may refer to drugsubstance and/or drug product, exemplary drug substances and drugproducts that may be assayed are as follows.

Exemplary drug substance for CTLA4-Ig molecules comprising SEQ ID NO:s2, 5, 6, 7, 8, 9, 10 or 18 is CLTA4-Ig protein at a concentration of 50mg/ml, in a buffered aqueous solution (25 mM sodium phosphate, 50 mMsodium chloride, pH of 7.5).

Exemplary drug product for CTLA4-Ig molecules comprising SEQ ID NO:s 2,5, 6, 7, 8, 9, 10 or 18 is, 250 mg lyophilized CTLA4-Ig protein, 500 mgmaltose, 17.2 mg monobasic sodium phosphate, and 14.6 mg sodiumchloride, pH 7.0-8.0; or

Composition of lyophilized CTLA4-Ig protein (250 mg/vial) drug productAmount Component (mg/vial)^(a) CTLA4-Ig protein 262.5 Maltosemonohydrate 525 Sodium phosphate monobasic, monohydrate^(b) 18.1 Sodiumchloride^(b) 15.3 Hydrochloric Acid Adjust to pH 7.5 Sodium hydroxideAdjust to pH 7.5buffered aqueous solution (25 mM sodium phosphate, 10 mM sodiumchloride, pH of 7.5).

Exemplary drug product for CTLA4Ig molecules comprising SEQ ID NO:s 4,11, 12, 13, 14, 15, 16, or 24:

Composition of lyophilized CLTA4^(A29YL104E)-Ig 100 mg/vial drug productAmount/Vial Component (mg) CLTA4^(A29YL104E)-Ig 110 Sucrose 220 SodiumPhosphate Monobasic Monohydrate 15.18 Sodium Chloride 2.55 1N SodiumHydroxide Adjust to pH 7.5 1N Hydrochloric Acid Adjust to pH 7.5

As used herein, the terms “culture medium” and “cell culture medium” and“feed medium” and “fermentation medium” refer to a nutrient solutionsused for growing and or maintaining cells, especially mammalian cells.Without limitation, these solutions ordinarily provide at least onecomponent from one or more of the following categories: (1) an energysource, usually in the form of a carbohydrate such as glucose; (2) allessential amino acids, and usually the basic set of twenty amino acidsplus cysteine; (3) vitamins and/or other organic compounds required atlow concentrations; (4) free fatty acids or lipids, for example linoleicacid; and (5) trace elements, where trace elements are defined asinorganic compounds or naturally occurring elements that are typicallyrequired at very low concentrations, usually in the micromolar range.The nutrient solution can be supplemented electively with one or morecomponents from any of the following categories: (1) hormones and othergrowth factors such as, serum, insulin, transferrin, and epidermalgrowth factor; (2) salts, for example, magnesium, calcium, andphosphate; (3) buffers, such as HEPES; (4) nucleosides and bases suchas, adenosine, thymidine, and hypoxanthine; (5) protein and tissuehydrolysates, for example peptone or peptone mixtures which can beobtained from purified gelatin, plant material, or animal byproducts;(6) antibiotics, such as gentamycin; (7) cell protective agents, forexample pluronic polyol; and (8) galactose.

The term “inoculation” as used herein refers to the addition of cells toculture medium to start the culture.

The term “growth phase” of the cell culture as used herein refers to theperiod of exponential cell growth (for example, the log phase) wherecells are primarily dividing rapidly. During this phase, the rate ofincrease in the density of viable cells is higher than at any other timepoint.

As used herein, the term “production phase” of the cell culture refersto the period of time during which cell growth is stationary or ismaintained at a near constant level. The density of viable cells remainsapproximately constant over a given period of time. Logarithmic cellgrowth has terminated and protein production is the primary activityduring the production phase. The medium at this time is generallysupplemented to support continued protein production and to achieve thedesired glycoprotein product.

As used herein, the terms “expression” or “expresses” are used to referto transcription and translation occurring within a cell. The level ofexpression of a product gene in a host cell can be determined on thebasis of either the amount of corresponding mRNA that is present in thecell or the amount of the protein encoded by the product gene that isproduced by the cell, or both.

As used herein, “glycosylation” refers to the addition of complexoligosaccharide structures to a protein at specific sites within thepolypeptide chain. Glycosylation of proteins and the subsequentprocessing of the added carbohydrates can affect protein folding andstructure, protein stability, including protein half life, andfunctional properties of a protein. Protein glycosylation can be dividedinto two classes by virtue of the sequence context where themodification occurs, O-linked glycosylation and N-linked glycosylation.O-linked polysaccharides are linked to a hydroxyl group, usually to thehydroxyl group of either a serine or a threonine residue. O-glycans arenot added to every serine and threonine residue. O-linkedoligosaccharides are usually mono or biantennary, i.e. they comprise oneor at most two branches (antennas), and comprise from one to fourdifferent kinds of sugar residues, which are added one by one. N-linkedpolysaccharides are attached to the amide nitrogen of an asparagine.Only asparagines that are part of one of two tripeptide sequences,either asparagine-X-serine or asparagine-X-threonine (where X is anyamino acid except proline), are targets for glycosylation. N-linkedoligosaccharides can have from one to four branches referred to asmono-, bi-, tri-tetraantennary. The structures of and sugar residuesfound in N-and O-linked oligosaccharides are different. Despite thatdifference, the terminal residue on each branch of both N-and O-linkedpolysaccharide can be modified by a sialic acid molecule a modificationreferred as sialic acids capping. Sialic acid is a common name for afamily of unique nine-carbon monosaccharides, which can be linked toother oligosaccharides. Two family members are N-acetyl neuraminic acid,abbreviated as Neu5Ac or NANA, and N-glycolyl neuraminic acid,abbreviated as Neu5Gc or NGNA. The most common form of sialic acid inhumans is NANA. N-acetylneuraminic acid (NANA) is the primary sialicacid species present in CTLA4-Ig molecules. However, it should be notedthat minor but detectable levels of N glycolylneuraminic acid (NGNA) arealso present in CTLA4-Ig molecules. Furthermore, the method describedherein can be used to determine the number of moles of sialic acids forboth NANA and NGNA, and therefore levels of both NANA and NGNA aredetermined and reported for CTLA4-Ig molecules. N-and O-linkedoligosaccharides have different number of branches, which providedifferent number of positions to which sialic acid molecules can beattached. N-linked ologosaccharides can provide up to four attachmentpositions for sialic acids, while O-linked oligosaccharides can providetwo sites for sialic acid attachment.

As used herein, the term “large-scale process” can be usedinterchangeably with the term “industrial-scale process”. Furthermore,the term “culture vessel” can be used interchangeably with “bioreactor”,“reactor” and “tank”.

As used herein, the phrase “working solution(s)” refers to solutionsthat are used in a method. Non-limiting examples of working solutionsinclude buffers.

As used herein, “reference material” refers to a material that is usedas a standard in a method. For example, a reference material can be usedas a standard to which experimental samples will be compared.

The absence of a substance is contemplated where no lower limit isprovided with regard to a range of amounts of such substance.

As used herein, recited temperatures in reference to cell culture refersto the temperature setting on the instrument that regulates thetemperature of the bioreactor. Of course, the temperature of the liquidculture itself will adopt the temperature set on the instrumentregulating the temperature for the bioreactor. Where the temperaturerefers to a cell culture that is maintained on a shelf in an incubator,the temperature then refers to the shelf temperature of the incubator.

Non-Limiting Embodiments of the Invention:

The invention provides for compositions of CTLA4-Ig molecules andcompositions of mutant CTLA4-Ig molecules, such as CTLA4^(A29YL104E)-Ig.The invention provides for compositions with certain characteristics,such as certain amounts of bacterial endotoxin, bioburden, a pI within acertain range (or certain IEF bands within a pI of a certain range), acertain amount of monomer (single chain), dimer or high molecular weightspecies (such as tetramer), a certain tryptic peptide profile, a certainset of major bands on SDS-PAGE, a certain DNA content, an amount ofMCP-1 not exceeding a certain maximum, an amount of cell protein notexceeding a certain maximum, an amount of Triton X-100 not exceeding acertain maximum, an amount of Protein A not exceeding a certain maximum,a certain profile of N-linked carbohydrates, a certain aminomonosaccharide composition (GlcNac, GalNAc), a certain neutralmonosaccharide composition (galactose, fucose, mannose), a certainamount of B7 binding, a certain amount of activity in a IL-2 inhibitioncell assay, and /or a certain sialic acid composition (NANA, NGNA), ineach case where said certain amounts can be a range or ranges. Theinvention provides compositions with any one of the aforementionedcharacteristics, or more than one of the aforementioned characteristics,up to an including all of the aforementioned characteristics in any andall possible permutations or combinations. The invention includes allthe compositions of the invention in isolated or substantially purifiedform, or not in isolated or substantially purified form. The inventionprovides for compositions which are pharmaceutical compositions.

In one aspect, the invention is directed to a method for obtaining acomposition comprising an isolated population of CTLA4-Ig molecules froma liquid culture medium, the medium comprising an initial population ofCTLA4-Ig molecules, wherein (1) CTLA4-Ig molecules of the initialpopulation have one or more sialic acid residues, (2) the number ofsialic acid residues per CTLA4-Ig molecule varies within the initialpopulation, and (3) the initial population comprises CTLA4-Ig dimer andhigh molecular weight aggregate, and the method comprises (a) harvestingthe liquid culture medium from a culture of mammalian cells expressingCTLA4-Ig molecules; (b)separating the CTLA4-Ig molecules from cellularcomponents; (c) separating CTLA4-Ig dimers from CTLA4-Ig high molecularweight aggregates; and (d) separating the CTLA4-Ig molecules into two ormore fractions, wherein at least one fraction has a greater molar ratioof sialic acid to CTLA4-Ig molecules compared to at least one otherfraction, and wherein steps (b), (c) and (d) are carried outsimultaneously or in any order, so as to obtain said composition.

In one embodiment of the method of the invention, the harvesting in step(a) comprises obtaining a soluble fraction of the liquid culture. Inanother embodiment, steps (c) and (d) of the method comprise the use ofcolumn chromatography so as to obtain fractions of CTLA4-Ig moleculeshaving different sialic acid contents. In yet another embodiment, themethod further comprises use of column chromatography to reduce MCP-1content in the composition.

In some embodiments of the method of the invention, the CTLA4-Igmolecules comprise one or more polypeptides having SEQ ID NO:2, 5, 6, 7,8, 9, or 10. In other embodiments, the CTLA4-Ig molecules comprise oneor more polypeptides having SEQ ID NO:4, 11, 12, 13, 14, 15 or 16.

In some embodiment of the method of the invention, the fraction in (d)having the greater molar ratio of sialic acid to CTLA4-Ig moleculesexhibits an average molar ratio of sialic acid to CTLA4-Ig moleculesfrom about 8 to about 14. In specific embodiments, the average molarratio is from about 8 to about 11, from about 8 to about 10, or fromabout 8 to about 9.

The invention provides for a method for isolating CTLA4-Ig molecules,the method comprising: (i) obtaining a soluble fraction of a liquidculture comprising mammalian cells that produce composition comprisingCTLA4-Ig molecules; (ii) subjecting the soluble fraction to anionexchange chromatography to obtain an eluted composition comprisingCTLA4-Ig molecules; (iii) subjecting the composition of step (ii) tohydrophobic interaction chromatography so as to obtain an enrichedcomposition comprising CTLA4-Ig molecules; (iv) subjecting thecomposition of (iii) to affinity chromatography to obtain a furtherenriched composition comprising CTLA4-Ig molecules; and (v) subjectingthe composition of (iv) to anion exchange chromatography. In oneembodiment, the composition obtained in step (ii) is characterized by:(a) an average of 6.0-10.1 moles of NANA per mole of CTLA4Ig molecule;and (b) less than or equal to 25.7 area percent CTLA4-Ig high molecularweight species as determined by size exclusion chromatography andspectrophotometric detection. In another embodiment, the compositionobtained in step (iii) is characterized by: (a) an average of 6.8-11.4moles of NANA per mole of CTLA4Ig molecule; and (b) less than or equalto 2.5 area percent of CTLA4-Ig high molecular weight species asdetermined by size exclusion chromatography and spectrophotometricdetection. In a further embodiment, the composition obtained in step(iv) is characterized by: (a) an average of 8.0-11.0 moles of NANA permole of CTLA4-Ig molecule; and (b) less than or equal to 2.5 areapercent of CTLA4-Ig high molecular weight species. In anotherembodiment, the composition obtained in step (v) is characterized by:(a) an average of 8.0-11.9 moles of NANA per mole of CTLA4-Ig molecule;and (b) less than or equal to 2.0 area percent being CTLA4-Ig highmolecular weight species as determined by size exclusion chromatographyand spectrophotometric detection (SPD). In one embodiment, an example ofSPD can be at A 280 nm.

The present invention also provides a method for isolating a compositionof CTLA4-Ig molecules comprising: (i) obtaining a soluble fraction of aliquid culture comprising mammalian cells that produce CTLA4-Igmolecules, and, in any order, (ii) subjecting the soluble fraction toanion exchange chromatography so as to obtain an enriched and elutedcomposition comprising CTLA4-Ig molecules; (iii) subjecting the solublefraction to hydrophobic interaction chromatography so as to obtain anenriched and eluted composition comprising CTLA4-Ig molecules; (iv)subjecting the soluble fraction to affinity chromatography so as toobtain an enriched and eluted composition comprising CTLA4-Ig molecules;and (v) subjecting the soluble fraction to anion exchange chromatographyso as to obtain an eriched and eluted composition comprising CTLA4-Igmolecules. In another aspect, the present invention provides a methodfor isolating a composition comprising CTLA4-Ig molecules, the methodcomprising: (i) obtaining a soluble fraction of a liquid culturecomprising mammalian cells that produce CTLA4-Ig molecules; (ii)subjecting the soluble fraction to anion exchange chromatography toobtain an eluted composition comprising CTLA4-Ig molecules; (iii)subjecting the protein product of step (ii) to hydrophobic interactionchromatography so as to obtain an enriched composition comprisingCTLA4-Ig molecules; (iv) subjecting the protein product of (iii) toaffinity chromatography to obtain a further enriched compositioncomprising CTLA4-Ig molecules; and (v) subjecting the protein product of(iv) to anion exchange chromatography, so as to isolate a compositioncomprising CTLA4-Ig molecules.

In one embodiment, the composition comprising CTLA4-Ig moleculesobtained in step (ii) of the method is characterized by: (a) an averagemolar ratio of NANA to CTLA4Ig molecules of from 6.0 to 10.1, and (b)less than or equal to 2.5 area percent CTLA4-Ig high molecular weightspecies as determined by size exclusion chromatography andspectrophotometric detection. In another embodiment, the compositioncomprising CTLA4-Ig molecules obtained in step (iii) of the method ischaracterized in that in that (a) CTLA4-Ig high molecular weight speciesis less than about 2.5 area % as determined by size exclusionchromatography and spectrophotometric detection, (b) cellular protein isless than about 95 ng/ml, and (c) MCP-1 is less than about 5 ppm. In anadditional embodiment, the composition comprising CTLA4-Ig moleculesobtained in step (iii) of the method is characterized by: (a) an averagemolar ratio of NANA to CTLA4-Ig molecules of from 6.8 to 11.4, and (b)less than or equal to 2.5 area percent CTLA4-Ig high molecular weightspecies as determined by size exclusion chromatography andspectrophotometric detection. In a further embodiment, the compositioncomprising CTLA4-Ig molecules obtained in step (iv) of the method ischaracterized by: (a) an average molar ratio of NANA to CTLA4-Igmolecules of from 8.0 to 11.0, and (b) less than or equal to 2.5 areapercent CTLA4-Ig high molecular weight species as determined by sizeexclusion chromatography and spectrophotometric detection. In stillanother embodiment, the composition obtained in step (iii) of theinvention is characterized in that CTLA4-Ig high molecular weightspecies is less than 2.5% area percent as determined by size exclusionchromatography and spectrophotometric detection. In yet anotherembodiment, the protein composition comprising CTLA4-Ig molecules instep (v) of the method is characterized by: (a) an average molar ratioof NANA to CTLA4-Ig molecules of from 8.0 to 11.9, and (b) less than orequal to 2.0 area percent CTLA4-Ig high molecular weight species asdetermined by size exclusion chromatography and spectrophotometricdetection.

The invention also provides, in another aspect, a method for isolating acomposition of CTLA4-Ig molecules, comprising: (i) obtaining a solublefraction of a liquid culture comprising mammalian cells that produceCTLA4-Ig molecules, and, in any order, (ii) subjecting the solublefraction to anion exchange chromatography so as to obtain an enrichedand eluted composition comprising CTLA4-Ig molecules; (iii) subjectingthe soluble fraction to hydrophobic interaction chromatography so as toobtain an enriched and eluted composition comprising CTLA4-Ig molecules;(iv) subjecting the soluble fraction to affinity chromatography so as toobtain an enriched and eluted composition comprising CTLA4-Ig molecules;and (v) subjecting the soluble fraction to anion exchange chromatographyso as to obtain an enriched and eluted composition comprising CTLA4-Igmolecules, wherein the composition obtained in step (iii) ischaracterized in that the percentage of CTLA4-Ig high molecular weightspecies is less than about 2.5 area %, cellular protein is less than 95ng/ml, and MCP-1 is less than about 5 ppm.

In still another aspect, the invention provides a method for isolating acomposition of CTLA4-Ig molecules, the method comprising: (i) obtaininga soluble fraction of a liquid culture comprising mammalian cells thatproduce CTLA4-Ig molecules, and, in any order, (ii) subjecting thesoluble fraction to anion exchange chromatography so as to obtain anenriched and eluted composition comprising CTLA4-Ig molecules; (iii)subjecting the soluble fraction to hydrophobic interactionchromatography so as to obtain an enriched and eluted compositioncomprising CTLA4-Ig molecules; (iv) subjecting the soluble fraction toaffinity chromatography so as to obtain an enriched and elutedcomposition comprising CTLA4-Ig molecules; and (v) subjecting thesoluble fraction to anion exchange chromatography so as to obtain anenriched and eluted composition comprising CTLA4-Ig molecules, whereinthe composition obtained in step (iii) is characterized in that thepercentage of CTLA4-Ig high molecular weight species is less than about2.5 area %, cellular protein is less than 95 ng/ml, MCP-1 is less thanabout 5 ppm, and the average molar ratio of NANA to CTLA4-Ig moleculesis of from about 8.0 to about 12.

In one embodiment, the anion exchange chromatography of step (ii) of themethod is carried out using a wash buffer comprising about 75 mM HEPES,and about 360 mM NaCl, and having a pH of about 8.0. In anotherembodiment, the anion exchange chromatography of step (ii) of theinvention is carried out using an elution buffer comprising about 25 mMHEPES, and about 850 mM NaCl, and having a pH of about 7.0. In anadditional embodiment, the hydrophobic interaction chromatography ofstep (iii) of the method is carried out using a single wash buffercomprising about 25 mM HEPES, and about 850 mM NaCl, and having a pH ofabout 7.0. In a further embodiment, the affinity chromatography of step(iv) of the method is carried out using a wash buffer comprising about25 mM Tris, and about 250 mM NaCl, and having a pH of about 8.0. Instill another embodiment, the affinity chromatography of step (iv) ofthe method is carried out using an elution buffer comprising about 100mM glycine and having a pH of about 3.5. In yet another embodiment, theanion exchange chromatography of step (v) of the method is carried outusing a wash buffer comprising about 25 mM HEPES, and from about 120 mMNaCl to about 130 mM NaCl, and having a pH of about 8.0. In stillanother embodiment, the anion exchange chromatography of step (v) of themethod is carried out using an elution buffer comprising about 25 mMHEPES, and about 200 mM NaCl, and having a pH of about 8.0. In yetanother embodiment, the anion exchange chromatography of step (ii) ofthe method is carried out using a column having an anion exchange resincomprising a primary, secondary, tertiary, or quarternary aminefunctional group. In a specific embodiment, the resin comprises aquarternary amine functional group. In still another embodiment, thehydrophobic interaction chromatography of step (iii) of the method iscarried out using a hydrophobic interaction resin comprising a phenyl,an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functional group.In a specific embodiment, the functional group comprises a phenylfunctional group. In still another embodiment, the affinitychromatography of step (iv) of the method is carried out using anaffinity chromatography resin comprising Protein A.

In yet another aspect, the invention provides a method for preparing acomposition comprising CTLA4-Ig molecules, comprising purifying CTLA4-Igmolecules from a liquid cell culture, wherein the purified CTLA4-Igcomposition comprises (a) a pharmaceutically acceptable amount of MCP-1per mg of CTLA4-Ig molecules, and (b) less than 2.5 area % of CTLA4-Ighigh molecular weight species as determined by size exclusionchromatography and spectrophotometric detection. In one embodiment, thepharmaceutically acceptable amount of MCP-1 comprises from about 40 toabout 0.5 ng/mg of CTLA4-Ig molecules. In another embodiment, thepharmaceutically acceptable amount of MCP-1 comprises from about 35 toabout 0.5 ng/mg of CTLA4-Ig molecules. In an additional embodiment, thepharmaceutically acceptable amount of MCP-1 comprises from about 10 toabout 0.5 ng/mg of CTLA4-Ig molecules. In a further embodiment, theaffinity chromatography of step (iv) of the method is carried out usinga column comprising a resin capable of reducing MCP-1 in the elutedprotein product. In still another embodiment, the hydrophobicinteraction chromatography of step (iii) of the method is carried outusing a hydrophobic interaction resin, wherein the resin is capable of(a) separating CTLA4-Ig dimers from CTLA4-Ig high molecular weightspecies; (b) increasing sialic acid content of the eluted CTLA4-Igmolecules; or (c) both (a) and (b). In yet another embodiment, the anionexchange chromatography of step (ii) or step (iv), or both, is carriedout using an anion exchange resin, wherein the resin is capable of (a)decreasing the CTLA4-Ig high molecular weight aggregate content of theeluted composition; (b) increasing the sialic content of the elutedcomposition; or (c) both (a) and (b).

In another aspect, the invention provides a method for isolating acomposition comprising CTLA4-Ig molecules, the method comprising: (i)obtaining a soluble fraction of a liquid culture comprising mammaliancells that produce CTLA4-Ig molecules, and in any order; (ii) subjectingthe soluble fraction to affinity chromatography so as to obtain aneluted composition comprising CTLA4-Ig molecules; (iii) subjecting thesoluble fraction to anion exchange chromatography so as to obtain aneluted and enriched composition comprising CTLA4-Ig molecules; and (iv)subjecting the soluble fraction to hydrophobic interactionchromatography so as to obtain an eluted and enriched compositioncomprising CTLA4-Ig molecules. In one embodiment, the affinitychromatography step is performed first. In another embodiment, theaffinity chromatography of step (ii) of the method is carried out usinga resin comprising Protein A. In an additional embodiment, the affinitychromatography of step (ii) is carried out using an elution buffercomprising guanidine. In a further embodiment, the affinitychromatography of step (ii) is carried out using an elution buffercomprising urea. In yet another embodiment, the affinity chromatographyof step (ii) results in an increase in CTLA4-Ig dimers in the elutedcomposition comprising CTLA4-Ig molecules.

In yet another aspect, the invention provides a method for isolatingcomposition comprising CTLA4-Ig molecules from liquid harvested from amammalian cell culture, wherein the cells produce CTLA4-Ig molecules,the method comprising: (i) obtaining a soluble fraction of the harvestedliquid; (ii) subjecting the soluble fraction to affinity chromatographyto obtain an eluted composition comprising CTLA4-Ig molecules; (iii)subjecting the composition of step (ii) to anion exchange chromatographyso as to obtain an eluted and enriched composition comprising CTLA4-Igmolecules; and (iv) subjecting the composition from step (iii) tohydrophobic interaction chromatography to obtain a further enrichedcomposition comprising CTLA4-Ig molecules. In one embodiment, thecomposition obtained in step (iv) of the method is characterized in thatthe percentage of high molecular weight species is less than about 2.5area % as determined by size exclusion chromatography andspectrophotometric detection, and the percentage of cellular protein isless than about 95 ng/ml, and the percentage of MCP-1 is less than about5 ppm. In another embodiment, the anion exchange chromatography of step(iii) is carried out using a wash buffer comprising about 50 mM HEPES,and about 135 mM NaCl, and having a pH of about 7. In an additionalembodiment, the anion exchange chromatography of step (iii) is carriedout using an elution buffer comprising about 50 mM HEPES, and about 200mM NaCl, and having a pH of about 7. In a specific embodiment, thehydrophobic interaction chromatography of step (iii) is carried outusing a hydrophobic interaction resin comprising a phenyl, an octyl, apropyl, an alkoxy, a butyl, or an isoamyl functional group. In a furtherembodiment, the hydrophobic interaction chromatography of step (iv) iscarried out using a wash buffer comprising about 50 mM HEPES, and about1.2 M (NH₄)₂SO₄, and having a pH of about 7. In still anotherembodiment, the affinity chromatography of step (ii) is carried outusing a wash buffer comprising about 25 mM NaH₂PO₄, and about 150 mMNaCl, and having a pH of about 7.5. In yet another embodiment, theaffinity chromatography of step (ii) is carried out using an elutionbuffer comprising about 250 mM glycine and having a pH of about 3. Inanother embodiment, the anion exchange chromatography of step (iii) iscarried out using a column having an anion exchange resin comprising aprimary, secondary, tertiary, or quarternary amine functional group. Ina specific embodiment, the resin comprises a quarternary aminefunctional group.

In one embodiment, the hydrophobic interaction chromatography of step(iii) is carried out using a hydrophobic interaction resin comprising aphenyl, an octyl, a propyl, an alkoxy, a butyl, or an isoamyl functionalgroup. In one embodiment, the functional group comprises a phenylfunctional group. In one embodiment, the affinity chromatography of step(ii) is carried out using a resin comprising Protein A. The inventionprovides for a composition comprising CTLA4-Ig molecules obtained by anyof the methods of the invention. In one embodiment, the compositioncomprises one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9 or10. In one embodiment, the composition comprises one or morepolypeptides having SEQ ID NO:4, 11, 12, 13, 14, 15 or 16. The inventionprovides for a CTLA4-Ig expression plasmid having the nucleic acidsequence of SEQ ID NO:17. The invention provides for a substantiallypurified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Ig proteinof from about 5.5 to about 18. The invention provides for asubstantially purified composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules have an average molar ratio of sialicacid to CTLA4-Ig molecules of from about 5.5 to about 9.5.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of from about 5to about 10. The invention provides for a substantially purifiedcomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Igmolecules of from about 6 to about 18. The invention provides for asubstantially purified composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules have an average molar ratio of sialicacid to CTLA4-Ig molecules of from about 8 to about 18.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of from about 8to about 12. The invention provides for a substantially purifiedcomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Igmolecules of from about 8 to about 11. The invention provides for asubstantially purified composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules have an average molar ratio of sialicacid to CTLA4-Ig molecules of from about 7 to about 12.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of from about 7to about 11. The invention provides for a substantially purifiedcomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Igmolecules of from about 11 to about 18. The invention provides for asubstantially purified composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules have an average molar ratio of sialicacid to CTLA4-Ig molecules of from about 12 to about 18.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of from about13 to about 18. The invention provides for a substantially purifiedcomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Igmolecules of from about 14 to about 18. The invention provides for asubstantially purified composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules have an average molar ratio of sialicacid to CTLA4-Ig molecules of from about 15 to about 17.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of about 16.The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of about 10.The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of sialic acid to CTLA4-Ig molecules of about 6. Inone embodiment, the sialic acid is N-acetyl neuraminic acid (NANA). Theinvention provides for a substantially purified composition comprisingCTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molarratio of NANA to CTLA4-Ig molecules of from about 8 to about 12. Theinvention provides for a substantially purified composition comprisingCTLA4-Ig molecules, wherein the CTLA4-Ig molecules have an average molarratio of N-glycolyl neuraminic acid (NGNA) to CTLA4-Ig molecules of lessthan or equal to about 1.5.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules have anaverage molar ratio of NGNA to CTLA4-Ig molecules of from about 0.5 toabout 1.5. The invention provides for a substantially purifiedcomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of NGNA to CTLA4-Ig molecules offrom about 1.0 to about 1.5. The invention provides for a substantiallypurified composition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules have an average molar ratio of sialic acid to CTLA4-Igmolecules of from about 6 to about 18.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of sialic acid per mole ofCTLA4-Ig molecules of from about 6 to about 12.

The invention provides for a substantially purified compositioncomprising CTLA4-Ig molecules, wherein each polypeptide of the moleculecomprises the sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, andwherein the CTLA4-Ig molecules are characterized by an average molarratio of sialic acid per mole of CTLA4-Ig molecules of from about 5.5 toabout 9.5. In one embodiment, the molar ratio of sialic acid per mole ofCTLA4-Ig molecules is determined by acid hydrolysis and HPLC. In oneembodiment, the CTLA4-Ig molecules comprise one or more polypeptideshaving SEQ ID NO:2, 5, 6, 7, 8, 9 or 10.

In one embodiment, the CTLA4-Ig molecules comprise one or morepolypeptides having SEQ ID NO:4, 11, 12, 13, 14, 15 or 16. The inventionprovides for a substantially purified composition comprising CTLA4-Igmolecules, wherein greater than or equal to 95% of the CTLA4-Igmolecules are CTLA4-Ig dimers. In one embodiment, greater than or equalto 98% of the CTLA4-Ig molecules are CTLA4-Ig dimers. In one embodiment,greater than or equal to 99% of the CTLA4-Ig molecules are CTLA4-Igdimers.

In one embodiment, greater than or equal to 99.5% of the CTLA4-Igmolecules are CTLA4-Ig dimers. In one embodiment, from about 95% toabout 99.5% of the CTLA4-Ig molecules are CTLA4-Ig dimers and about 0.5area percent to about 5 area percent of the molecules are CTLA4-Ig highmolecular weight species as determined by size exclusion chromatographyand spectrophotometric detection. In one embodiment, about 98.6% of themolecules are CTLA4-Ig dimers and about 1.2 area percent of themolecules are CTLA4-Ig high molecular weight species and about less than0.7 area percent of the molecules are CTLA4-Ig monomers as determined bysize exclusion chromatography and spectrophotometric detection. In oneembodiment, about less then about 0.3% of the molecules are multimerscomprising five or more CTLA4-Ig monomers. The invention provides for acomposition consisting essentially of CTLA4-Ig dimers. The inventionprovides for a composition consisting essentially of CTLA4-Ig molecules,wherein the population is substantially free of CTLA4-Ig monomers. Theinvention provides for a composition consisting essentially of CTLA4-Igmolecules, wherein the population is substantially free of CTLA4-Ig highmolecular weight species. The invention provides for a compositionconsisting essentially of CTLA4-Ig monomers substantially free ofCTLA4-Ig dimers and high molecular weight species. In one embodiment,each monomer of each CTLA4-Ig dimer has at least 3 sialic acid groups.In one embodiment, each monomer of each CTLA4-Ig dimer has at least 2.5sialic acid groups. In one embodiment, each monomer of each CTLA4-Igdimer has from at least 3 sialic acid groups to at least 8 sialic acidgroups.

In one embodiment, each monomer of each CTLA4-Ig dimer has from at least2.5 sialic acid groups to at least 5 sialic acid groups. In oneembodiment, each dimer comprises two CTLA4-Ig polypeptides, wherein eachpolypeptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS:5-16. In one embodiment, the compositioncomprises one or more polypeptides having SEQ ID NO:2, 5, 6, 7, 8, 9 or10. In one embodiment, the composition comprises one or morepolypeptides having SEQ ID NO:4, 11, 12, 13, 14, 15 or 16. The inventionprovides for an isolated composition comprising CTLA4-Ig tetramers,which is substantially free of CTLA4-Ig dimers. The invention providesfor an isolated composition comprising CTLA4-Ig tetramers which issubstantially free of CTLA4-Ig monomers. In one embodiment, thecomposition exists as an amount that is greater than about 100 grams. Inone embodiment, each tetramer comprises two pairs of CTLA4-Igpolypeptides, wherein each polypeptide has an amino acid sequenceselected from the group consisting of SEQ ID NOS:5-10. In oneembodiment, each tetramer comprises two pairs of CTLA4-Ig polypeptides,wherein each polypeptide has an amino acid sequence selected from thegroup consisting of SEQ ID NOS:11-16. In one embodiment, each tetrameris capable of binding to a CD80 or CD86. The invention provides for apharmaceutically acceptable composition comprising CTLA4-Ig molecules,wherein the composition is substantially free of MCP-1. The inventionprovides for a pharmaceutically acceptable composition comprisingCTLA4-Ig molecules, wherein the composition comprises no more than about25 ppm MCP-1. In one embodiment, the composition comprises no more than10 ppm MCP-1. In one embodiment, the composition comprises from about0.2 ng/ml MCP-1 to about 10 ng/ml of MCP-1. In one embodiment, theinvention provides for a pharmaceutically acceptable compositioncomprising CTLA4-Ig molecules, wherein the composition comprises (a)from about 0.2 ng/ml MCP-1 to about 10 ng/ml of MCP-1 and (b) no morethan 25 ng/ml of CHO protein or no more than 10 ng/ml of CHO protein. Inone embodiment, the composition comprises no more than about 20 pg/ml ofDNA.

The invention provides for an isolated composition comprising CTLA4-Igmolecules, wherein, when administered to a subject at an intravenousdose of about 10 mg/kg, the CTLA4-Ig molecules are capable ofexhibiting: an area under the curve (AUC) of about 44400 μg/ml; a volumeof distribution of about 0.09 L/kg; a peak concentration (Cmax) of about292 μg/ml; and a clearance rate of about 0.23 ml/h/kg. The inventionprovides for an isolated composition comprising CTLA4-Ig molecules,wherein the composition comprises dominant isoforms of CTLA4-Igmolecules visualizable on an isoelectric focusing gel which have anisoelectric point, pI, less than or equal to 5.1±0.2 as determined byisoelectric focusing. In one embodiment, the average pI of thecomposition increases after neuraminidase treatment. In one embodiment,at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point lessthan or equal to about 5.1±0.2 as determined by isoelectric focusing. Inone embodiment, at least 70% of the CTLA4-Ig molecules exhibit anisoelectric point less than or equal to about 5.1±0.2 as determined byisoelectric focusing. In one embodiment, at least 90% of the CTLA4-Igmolecules exhibit an isoelectric point less than or equal to about5.1±0.2 as determined by isoelectric focusing. The invention providesfor an isolated composition comprising CTLA4-Ig molecules having a pI offrom about 3.0±0.2 to about 5.0±0.2. The invention provides for anisolated composition comprising CTLA4-Ig molecules having a pI fromabout 4.3±0.2 to about 5.0±0.2.

The invention provides for an isolated composition comprising CTLA4-Igmolecules having a pI of about 3.3±0.2 to about 4.7±0.2. In oneembodiment, the composition is substantially purified. The inventionprovides for a method for preparing a composition, the compositioncomprising a CTLA4-Ig molecule with a pI of from about 3.0±0.2 to about5.0±0.2, the method comprising: (a) subjecting a mixture of CTLA4-Igmolecules to isoelectric focusing gel electrophoresis, wherein a singleband on the gel represents a population of CTLA4-Ig molecules with aparticular pI, and (b) isolating the population of CTLA4-Ig moleculeshaving a pI of from about 3.0±0.2 to about 5.0±0.2 so as to prepare thecomposition. The invention provides for an isolated compositioncomprising CTLA4-Ig molecules, wherein the composition comprisesdominant isoforms visualizable on an isoelectric focusing gel which havean isoelectric point, pI, less than or equal to 5.5±0.2 as determined byisoelectric focusing. In one embodiment, the average pI of thecomposition increases after neuraminidase treatment. In one embodiment,at least 40% of the CTLA4-Ig molecules exhibit an isoelectric point lessthan or equal to about 5.3±0.2 as determined by isoelectric focusing. Inone embodiment, at least 70% of the CTLA4-Ig molecules exhibit anisoelectric point less than or equal to about 5.3±0.2 as determined byisoelectric focusing. In one embodiment, at least 90% of the CTLA4-Igmolecules exhibit an isoelectric point less than or equal to about5.3±0.2 as determined by isoelectric focusing. The invention providesfor an isolated composition comprising CTLA4-Ig molecules having a pI offrom about 3.0±0.2 to about 5.2±0.2.

The invention provides for an isolated composition comprising CTLA4-Igmolecules having a pI from about 4.5±0.2 to about 5.2±0.2. The inventionprovides for an isolated composition comprising CTLA4-Ig moleculeshaving a pI of about 4.7±0.2 to about 5.1±0.2. In one embodiment, thecomposition is substantially purified.

The invention provides for a method for preparing a composition, thecomposition comprising CTLA4-Ig molecules with a pI of from about2.0±0.2 to about 5.2±0.2, the method comprising: (a) subjecting amixture of CTLA4-Ig molecules to isoelectric focusing gelelectrophoresis, wherein a single band on the gel represents apopulation of CTLA4-Ig molecules with a particular pI, and (b) isolatingthe population of CTLA4-Ig molecules having a pI of from about 3.0±0.2to about 5.2±0.2 so as to prepare the composition. The inventionprovides for a composition comprising CTLA4-Ig molecules, wherein theCTLA4-Ig molecules are characterized by an average molar ratio of GlcNActo CTLA4-Ig molecules of from about 17 to about 28. The inventionprovides for a composition comprising CTLA4-Ig molecules, wherein theCTLA4-Ig molecules are characterized by an average molar ratio of GlcNActo CTLA4-Ig molecules of from about 17 to about 25. The inventionprovides for a composition comprising CTLA4-Ig molecules, wherein theCTLA4-Ig molecules are characterized by an average molar ratio of GlcNActo CTLA4-Ig molecules of from about 15 to about 35.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein each polypeptide of the molecule comprises the sequence of SEQID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of GlcNAc to CTLA4-Ig moleculesof from about 24 to about 28. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of GalNAc to CTLA4-Ig moleculesof from about 1.7 to about 3.6. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein each polypeptide of the moleculecomprises the sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, andwherein the CTLA4-Ig molecules are characterized by an average molarratio of GalNAc to CTLA4-Ig molecules of from about 2.7 to about 3.6.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of galactose to CTLA4-Ig molecules of from about 8 to about 17.The invention provides for a composition comprising CTLA4-Ig molecules,wherein each polypeptide of the molecule comprises the sequence of SEQID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of galactose to CTLA4-Igmolecules of from about 11 to about 13. The invention provides for acomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules are characterized by an average molar ratio of fucose toCTLA4-Ig molecules of from about 3.5 to about 8.3.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein each polypeptide of the molecule comprises the sequence of SEQID NO:11, 12, 13, 14, 15 or 16, and wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of fucose to CTLA4-Ig moleculesof from about 6.4 to about 7.0. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of mannose to CTLA4-Ig moleculesof from about 7.7 to about 22. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein each polypeptide of the moleculecomprises the sequence of SEQ ID NO:11, 12, 13, 14, 15 or 16, andwherein the CTLA4-Ig molecules are characterized by an average molarratio of mannose to CTLA4-Ig molecules of from about 14 to about 16.

In one embodiment, the molar ratio of GlcNAc to CTLA4-Ig molecules isdetermined by capillary electrophoresis. In one embodiment, the molarratio of GalNAc to CTLA4-Ig molecules is determined by capillaryelectrophoresis. In one embodiment, the molar ratio of galactose toCTLA4-Ig molecules is determined by capillary electrophoresis.

In one embodiment, the molar ratio of fucose to CTLA4-Ig molecules isdetermined by capillary electrophoresis. In one embodiment, the molarratio of mannose to CTLA4-Ig molecules is determined by capillaryelectrophoresis. In one embodiment, the CTLA4-Ig molecules are obtainedby enzymatic attachment of one or more carbohydrates to the molecule.The invention provides for a composition comprising CTLA4-Ig molecules,wherein the molecules comprise carbohydrate residues attached to themolecules enzymatically in vitro. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 toabout 35; and (b) an average molar ratio of sialic acid to CTLA4-Igmolecules from about 6 to about 12. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc to CTLA4-Ig molecules from about 15 toabout 35; (b) an average molar ratio of GalNAc to CTLA4-Ig moleculesfrom about 1.7 to about 3.6; and (c) an average molar ratio of sialicacid to CTLA4-Ig molecules from about 6 to about 12. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules fromabout 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Igmolecules from about 1.7 to about 3.6; (c) an average molar ratio ofgalactose to CTLA4-Ig molecules from about 8 to about 17; and (d) anaverage molar ratio of sialic acid to CTLA4-Ig molecules from about 6 toabout 12. The invention provides for a composition comprising CTLA4-Igmolecules characterized by: (a) an average molar ratio of GlcNAc toCTLA4-Ig molecules from about 15 to about 35; (b) an average molar ratioof GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c) anaverage molar ratio of galactose to CTLA4-Ig molecules from about 8 toabout 17; (d) an average molar ratio of fucose to CTLA4-Ig moleculesfrom about 3.5 to about 8.3; and (e) an average molar ratio of sialicacid to CTLA4-Ig molecules from about 6 to about 12. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules fromabout 15 to about 35; (b) an average molar ratio of GalNAc to CTLA4-Igmolecules from about 1.7 to about 3.6; (c) an average molar ratio ofgalactose to CTLA4-Ig molecules from about 8 to about 17; (d) an averagemolar ratio of fucose to CTLA4-Ig molecules from about 3.5 to about 8.3;(e) an average molar ratio of mannose to CTLA4-Ig molecules from about7.2 to about 22; and (f) an average molar ratio of sialic acid toCTLA4-Ig molecules from about 6 to about 12. The invention provides fora composition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 toabout 28; and (b) an average molar ratio of sialic acid to CTLA4-Igmolecules from about 5.5 to about 9.5. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc to CTLA4-Ig molecules from about 24 toabout 28; (b) an average molar ratio of GalNAc to CTLA4-Ig moleculesfrom about 2.7 to about 3.6; and (c) an average molar ratio of sialicacid to CTLA4-Ig molecules from about 5.5 to about 9.5. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of GlcNAc to CTLA4-Ig molecules fromabout 24 to about 28; (b) an average molar ratio of GalNAc to CTLA4-Igmolecules from about 2.7 to about 3.6; (c) an average molar ratio ofgalactose to CTLA4-Ig molecules from about 11 to about 13; and (d) anaverage molar ratio of sialic acid to CTLA4-Ig molecules from about 5.5to about 9.5. The invention provides for a composition comprisingCTLA4-Ig molecules characterized by: (a) an average molar ratio ofGlcNAc to CTLA4-Ig molecules from about 24 to about 28; (b) an averagemolar ratio of GalNAc to CTLA4-Ig molecules from about 2.7 to about 3.6;(c) an average molar ratio of galactose to CTLA4-Ig molecules from about11 to about 13; (d) an average molar ratio of fucose to CTLA4-Igmolecules from about 6.4 to about 7.0; and (e) an average molar ratio ofsialic acid to CTLA4-Ig molecules from about 5.5 to about 9.5. Theinvention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of GlcNAc to CTLA4-Igmolecules from about 24 to about 28; (b) an average molar ratio ofGalNAc to CTLA4-Ig molecules from about 2.7 to about 3.6; (c) an averagemolar ratio of galactose to CTLA4-Ig molecules from about 11 to about13; (d) an average molar ratio of fucose to CTLA4-Ig molecules fromabout 6.4 to about 7.0; (e) an average molar ratio of mannose toCTLA4-Ig protein from about 14 to about 16; and (f) an average molarratio of sialic acid to CTLA4-Ig protein from about 5.5 to about 9.5.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are glycosylated at an asparagine aminoacid residue at position 102 of SEQ ID NO:2 or 4, an asparagine aminoacid residue at position 134 of SEQ ID NO:2 or 4, an asparagine aminoacid residue at position 233 of SEQ ID NO:2 or 4, a serine amino acidresidue at position 155 of SEQ ID NO:2 or 4, or a serine amino acidresidue at position 165 of SEQ ID NO:2 or 4. The invention provides fora composition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules are glycosylated, and wherein at least about 2% of total massof glycosylation is O-linked glycosylation.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the composition exhibits an NGNA chromatogram peak of about9.6±0.3 and an NANA chromatogram peak of about 10.5±0.3. The inventionprovides for a composition comprising CTLA4-Ig molecules, wherein theCTLA4-Ig molecules exhibit a carbohydrate profile substantially the sameas FIG. 67. The invention provides for a composition comprising CTLA4-Igmolecules, wherein the CTLA-Ig molecules exhibit a carbohydrate profileas shown in FIG. 67. The invention provides for a composition consistingessentially of CTLA4-Ig molecules, wherein the CTLA4-Ig moleculesexhibit a carbohydrate profile of Domains I-IV, wherein Domain Icomprises peaks which represent a-sialylated oligosaccharides, Domain IIcomprises peaks which represent mono-sialylated oligosaccharides, DomainIII comprises peaks which represent di-sialylated oligosaccharides,Domain IV comprises peaks which represent tri-sialylatedoligosaccharides, and Domain V comprises peaks which representtetra-sialyated oligosaccharides, and wherein the profile is achromatogram of oligosaccharides released from CTLA4-Ig. In oneembodiment, the difference in retention times of N-linkedoligosaccharides between a first peak in Domain I and a main peak inDomain II is from about 10 to about 12 minutes. In one embodiment, thedifference in retention times of N-linked oligosaccharides between afirst peak in Domain I and a main peak in Domain II is from about 11 toabout 13 minutes. In one embodiment, glycosylation of Domains III and IVcomprises about 25% to about 36% of N-linked glycosylation as measuredby HPAEC. In one embodiment, glycosylation of Domain I comprises about24.5% to about 35.2% of N-linked glycosylation as measured by HPAEC. Inone embodiment, glycosylation of Domain II comprises about 26.3% toabout 34.1% of N-linked glycosylation as measured by HPAEC. In oneembodiment, glycosylation of Domain III comprises about 21.9% to about31.5% of N-linked glycosylation as measured by HPAEC. In one embodiment,glycosylation of Domain IV and Domain V comprises about 7.9% to about18.6% of N-linked glycosylation as measured by HPAEC.

In one embodiment: (a) Domain I exhibits an area percentage of at leastabout 31; (b) Domain II exhibits an area percentage of at least about33; (c) Domain III exhibits an area percentage of at least about 24;(iv) Domain IV exhibits an area percentage of at least about 9.4, (v)Domain V exhibits an area percentage of at least about 67; or whereinthe area is measured from a chromatogram of oligosaccharides releasedfrom CTLA4-Ig.

In one embodiment: (a) Domain I exhibits at least about 5 peaks; (b)Domain II exhibits at least about 5 peaks; (c) Domain III exhibits atleast about 5 peaks; (d) Domain IV exhibits at least about 6 peaks, or(e) Domain V exhibits at least about 6 peaks, and wherein the peaks areexhibited on a chromatogram. A composition wherein Domain I exhibits atleast two peaks, wherein a first peak has a minimum area of about 4.5%and a maximum area of about 11.2%, and wherein a second peak has aminimum area of about 8.7% and a maximum of about 11.8%.

In one embodiment, Domain III and IV exhibit an area percentage of about25% to about 36% as measured by HPAEC. In one embodiment, Domain Iexhibits an area percentage of about 24.5% to about 35.2% as measured byHPAEC. In one embodiment, Domain II exhibits an area percentage of about26.3% to about 34.1% as measured by HPAEC. In one embodiment, Domain IIIexhibits an area percentage of about 21.9% to about 31.5% as measured byHPAEC. In one embodiment, Domain IV exhibits an area percentage of about7.9% to about 18.6% as measured by HPAEC.

The invention provides for a composition comprising CTLA4-Igpolypeptides, wherein: (a) about 80% of the polypeptides havebiantennary N-linked glycosylation; (b) about 14% of the polypeptideshave triantennary N-linked glycosylation; and (c) about 6% of thepolypeptides have tetraantennary N-linked glycosylation. In oneembodiment, the N-linked glycosylation is idetermined by high pH anionexchange chromatography with pulsed amperometric detection (HPEAC-PAD).The invention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of galactose to CTLA4-Igmolecules of from about 8 to about 17; and (b) an average molar ratio ofNANA to CTLA4-Ig molecules of from about 6 to about 12. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby:

(a) an average molar ratio of galactose to CTLA4-Ig molecules of fromabout 8 to about 17; (b) an average molar ratio of NANA to CTLA4-Igmolecules of from about 6 to about 12; and (c) a CTLA4-Ig high molecularweight species area percent of less than about 3% as determined by sizeexclusion chromatography and spectrophotometric detection. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of galactose to CTLA4-Ig molecules offrom about 8 to about 17; (b) an average molar ratio of NANA to CTLA4-Igmolecules of from about 6 to about 12; and (c) an average molar ratio ofNGNA to CTLA4-Ig molecules of less than or equal to about 1.5.

The invention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of galactose to CTLA4-Igmolecules of from about 8 to about 17; (b) an average molar ratio ofNANA to CTLA4-Ig molecules of from about 6 to about 12; (c) a CTLA4-Ighigh molecular weight aggregate content less than about 3 area percentas determined by size exclusion chromatography and spectrophotometricdetection; and (d) a carbohydrate profile substantially the same as thatof FIG. 67. The invention provides for a composition comprising CTLA4-Igmolecules characterized by: (a) an average molar ratio of galactose toCTLA4-Ig molecules of from about 8 to about 17; (b) an average molarratio of NANA to CTLA4-Ig molecules of from about 6 to about 12; (c) aCTLA4-Ig high molecular weight aggregate content less than about 3 areapercent as determined by size exclusion chromatography andspectrophotometric detection; and (d) a glycosylation content in DomainsIII, IV and V of at least about 29.8% to about 50.1% of N-linkedglycosylation as determined by HPAEC. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of galactose to CTLA4-Ig molecules of from about 8to about 17; (b) an average molar ratio of NANA to CTLA4-Ig molecules offrom about 6 to about 12; and (c) a CTLA4-Ig high molecular weightspecies of less than about 3 area percent as determined by sizeexclusion chromatography and spectrophotometric detection. In oneembodiment, the molecules are further characterized by an average molarratio of NANA to CTLA4-Ig molecules from about 8 to about 12.

In one embodiment, the molecules are further characterized by: (a) about80% biantennary N-linked glycosylation; (b) about 14% triantennaryN-linked glycosylation; and (c) about 6% tetraantennary N-linkedglycosylation. In one embodiment, the molecules further comprise anycombination of one or more of: (a) the amino acid sequence of SEQ IDNO:10 (methionine at amino acid position 27 and glycine at amino acidposition 382 of SEQ ID NO:2); (b) the amino acid sequence of SEQ ID NO:7(methionine at amino acid position 27 and lysine at amino acid position383 of SEQ ID NO:2); (c) the amino acid sequence of SEQ ID NO:9 (alanineat amino acid position 26 and glycine at amino acid position 382 of SEQID NO:2); and (d) the amino acid sequence of SEQ ID NO:6 (alanine atamino acid position 26 and lysine at amino acid position 383 of SEQ IDNO:2). In one embodiment, (a) about 90% of the molecules comprise theamino acid sequence of SEQ ID NO:2 beginning with the methionine atresidue 27; (b) about 10% of the molecules comprise the amino acidsequence of SEQ ID NO:2 beginning with the alanine at residue number 26;(c) about 4% of the molecules comprise the amino acid sequence of SEQ IDNO:2 ending with the lysine at residue number 383; and (d) about 96% ofthe molecules comprise the amino acid sequence of SEQ ID NO:2 endingwith the glycine at residue number 382. The invention provides for acomposition comprising CTLA4-Ig polypeptides, wherein: (a) about 80% ofthe polypeptides have biantennary N-linked glycosylation; (b) about 14%of the polypeptides have triantennary N-linked glycosylation; (c) about6% of the polypeptides have tetraantennary N-linked glycosylation;and(d) an average molar ratio of NGNA to CTLA4-Ig molecules of less thanor equal to 1.5. The invention provides for a composition comprisingCTLA4-Ig polypeptides, wherein: (a) about 80% of the polypeptides havebiantennary N-linked glycosylation; (b) about 14% of the polypeptideshave triantennary N-linked glycosylation; (c) about 6% of thepolypeptides have tetraantennary N-linked glycosylation; and(d) anaverage molar ratio of GlcNAc to CTLA4-Ig molecules of from about 15 toabout 35. The invention provides for a composition comprising CTLA4-Igpolypeptides, wherein: (a) about 80% of the polypeptides havebiantennary N-linked glycosylation; (b) about 14% of the polypeptideshave triantennary N-linked glycosylation; (c) about 6% of thepolypeptides have tetraantennary N-linked glycosylation; and(d) anaverage molar ratio of GalNAc to CTLA4-Ig molecules of from about 1.7 toabout 3.6. The invention provides for a composition comprising CTLA4-Igmolecules characterized by: (a) an average molar ratio of galactose toCTLA4-Ig molecules of from about 11 to about 13; and (b) an averagemolar ratio of sialic to CTLA4-Ig molecules of from about 5.5 to about9.5.

The invention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of galactose to CTLA4-Igmolecules of from about 11 to about 13; (b) an average molar ratio ofsialic acid to CTLA4-Ig molecules of from about 5.5 to about 9.5; and(c) a CTLA4-Ig high molecular weight species of less than about 5 areapercent as determined by size exclusion chromatography andspectrophotometric detection. The invention provides for a compositioncomprising CTLA4-Ig molecules characterized by: (a) an average molarratio of galactose to CTLA4-Ig molecules of from about 11 to about 13;

(b) an average molar ratio of sialic acid to CTLA4-Ig molecules of fromabout 5.5 to about 9.5; (c) a CTLA4-Ig high molecular weight speciescontent less than about 5 area percent as determined by size exclusionchromatography and spectrophotometric detection; and (d) a carbohydrateprofile substantially the same as that of FIG. 67. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of galactose to CTLA4-Ig molecules offrom about 11 to about 13; (b) an average molar ratio of sialic acid toCTLA4-Ig molecules of from 5.5 to about 9.5; (c) a CTLA4-Ig highmolecular weight species content less than about 5 area percent asdetermined by size exclusion chromatography and spectrophotometricdetection; and (d) a glycosylation content in Domains III, IV and V ofat least about 29.8% to about 50.1% of N-linked glycosylation asdetermined by HPAEC.

The invention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of galactose to CTLA4-Igmolecules of from about 11 to about 13; (b) an average molar ratio ofsialic acid to CTLA4-Ig molecules of from about 5.5 to about 9.5; and(c) a CTLA4-Ig high molecular weight species content less than about 5area percent as determined by size exclusion chromatography andspectrophotometric detection. In one embodiment, the molecules arefurther characterized by: (a) about 80% biantennary N-linkedglycosylation; (b) about 14% triantennary N-linked glycosylation; and(c) about 6% tetraantennary N-linked glycosylation. In anotherembodiment, the molecules further comprise any combination of one ormore of: (a) the amino acid sequence of SEQ ID NO:16 (methionine atamino acid position 27 and glycine at amino acid position 382 of SEQ IDNO:4); (b) the amino acid sequence of SEQ ID NO:13 (methionine at aminoacid position 27 and lysine at amino acid position 383 of SEQ ID NO:4);(c) the amino acid sequence of SEQ ID NO:15 (alanine at amino acidposition 26 and glycine at amino acid position 382 of SEQ ID NO:4); and(d) the amino acid sequence of SEQ ID NO:12 (alanine at amino acidposition 26 and lysine at amino acid position 383 of SEQ ID NO:4). Inanother embodiment, (a) about 90% of the molecules comprise the aminoacid sequence of SEQ ID NO:4 beginning with the methionine at residue27; (b) about 10% of the molecules comprise the amino acid sequence ofSEQ ID NO:4 beginning with the alanine at residue number 26; (c) about4% of the molecules comprise the amino acid sequence of SEQ ID NO:4ending with the lysine at residue number 383; and (d) about 96% of themolecules comprise the amino acid sequence of SEQ ID NO:4 ending withthe glycine at residue number 382. The invention provides for acomposition comprising CTLA4-Ig polypeptides, wherein:(a) about 80% ofthe polypeptides have biantennary N-linked glycosylation; (b) about 14%of the polypeptides have triantennary N-linked glycosylation; (c) about6% of the polypeptides have tetraantennary N-linked glycosylation;and(d) an average molar ratio of GlcNAc per mole of CTLA4-Ig protein offrom about 24 to about 28. The invention provides for a compositioncomprising CTLA4-Ig polypeptides, wherein:(a) about 80% of thepolypeptides have biantennary N-linked glycosylation; (b) about 14% ofthe polypeptides have triantennary N-linked glycosylation; (c) about 6%of the polypeptides have tetraantennary N-linked glycosylation; and(d)an average molar ratio of GalNAc to CTLA4-Ig molecules of from about 2.7to about 3.6. In another embodiment, the composition is a substantiallypurified composition. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein less than or equal to about 2.5%of the CTLA4-Ig molecules are oxidized. The invention provides for acomposition comprising CTLA4-Ig molecules, wherein less than or equal toabout 2.0% of the CTLA4-Ig molecules are deamidated. The inventionprovides for a composition comprising CTLA4-Ig dimer molecules, whereinat least 0.5% of the CTLA4-Ig dimer molecules are cysteinylated. In oneembodiment, at least 1.0% of the CTLA4-Ig dimer molecules arecysteinylated. The invention provides for a population of CTLA4-Igmolecules, wherein the population exhibits a mass spectrometry profilesubstantially the same as FIG. 63, 64 or 66. The invention provides fora population of CTLA4-Ig molecules, wherein the population exhibits acapillary electrophoresis profile substantially the same as FIG. 47.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the composition is characterized by: (a) an average molar ratioof GlcNAc to CTLA4-Ig molecules from about 15 to about 35; (b) anaverage molar ratio of GalNAc to CTLA4-Ig molecules from about 1.7 toabout 3.6; (c) an average molar ratio of galcatose to CTLA4-Ig moleculesfrom about 8 to about 17; (d) an average molar ratio of fucose toCTLA4-Ig molecules from about 3.5 to about 8.3; (e) an average molarratio of mannose to CTLA4-Ig molecules from about 7.2 to about 22; (f)an average molar ratio of sialic acid to CTLA4-Ig molecules from about 6to about 12; (g) a pI as determined from visualization on an isoelectricfocusing gel in a range from about 2.4±0.2 to about 5.0±0.2; (h) MCP-1of less than or equal to 3 ppm; (i) less than 2.5 area percent of highmolecular weight species as determined by size exclusion chromatographyand spectrophotometric detection; (j) less than 0.5 area percent ofmonomer as determined by size exclusion chromatography andspectrophotometric detection; (k) CTLA4-Ig polypeptides having an aminoacid at least 95% identical to any of SEQ ID NOS:5-10; (1) CTLA4-Igmolecules capable of binding to CD80 and CD86. The invention providesfor a composition comprising CTLA4-Ig molecules, wherein the populationof molecules is characterized by: (a) an average molar ratio of GlcNActo CTLA4-Ig molecules from about 15 to about 35; (b) an average molarratio of GalNAc to CTLA4-Ig molecules from about 1.7 to about 3.6; (c)an average molar ratio of galcatose to CTLA4-Ig molecules from about 8to about 17; (d) an average molar ratio of fucose to CTLA4-Ig moleculesfrom about 3.5 to about 8.3; (e) an average molar ratio of mannose toCTLA4-Ig molecules from about 7.2 to about 22; (f) an average molarratio of sialic acid to CTLA4-Ig molecules from about 6 to about 12; (g)a pI as determined from visualization on an isoelectric focusing gel ina range from about 3.4±0.2 to about 5.0±0.2; (h) MCP-1 of less than orequal to 5 ppm; (i) less than 2.5 area percent of high molecular weightspecies as determined by size exclusion chromatography andspectrophotometric detection; (j) less than 0.5 area percent of monomeras determined by size exclusion chromatography and spectrophotometricdetection; (k) CTLA4-Ig polypeptides having an amino acid at least 95%identical to any of SEQ ID NOS:5-10; (1) CTLA4-Ig molecules capable ofbinding to CD80 and CD86; or pharmaceutical equivalents thereof.

The invention provides for an isolated composition comprising CTLA4-Igmolecules having an incidence of immunogenicity of less than or equal to7.4%. In one embodiment, the incidence of immunogenicity is from about2.1% to about 7.4%. In one embodiment, the incidence of immunogenicityis less than or equal to 3.7%. In one embodiment, the incidence ofimmunogenicity is less than or equal to 3.0%. In one embodiment, theincidence of immunogenicity is from about 2.8% to about 3.0%. Theinvention provides for an isolated composition comprising CTLA4-Igmolecules, wherein, following administration of the composition tohumans, production of antibodies that bind to the CTLA4-Ig moleculesoccurs at an incidence in the humans of less than or equal to 7.4%. Inone embodiment, the incidence is from about 2.1% to about 7.4%. In oneembodiment, the incidence is less than or equal to 3.7%. In oneembodiment, the incidence is less than or equal to 3.0%. In oneembodiment, the incidence is from about 2.8% to about 3.0%. Theinvention provides for an isolated composition comprising CTLA4-Igmolecules, wherein, following administration of the composition tohumans, production of antibodies that bind to the CTLA4 portions of theCTLA4-Ig molecules occurs in the humans at an incidence of less than orequal to 4.9%. In one embodiment, the incidence is from about 0.5% toabout 4.9%. In one embodiment, the incidence is less than or equal to1.2%. In one embodiment, the incidence is less than or equal to 1.0%. Inone embodiment, the incidence is from about 0.9% to about 1.0%. In oneembodiment, the incidence is measured in an enzyme-lined immunosorbentassay (ELISA). In one embodiment, wherein the incidence is measured inan an electrochemoluminescence assay (ECL).

The invention provides for an isolated composition comprising CTLA4-Igmolecules, wherein, following administration of the composition tohumans, production of antibodies that neutralize the CTLA4-Ig moleculesoccurs at an incidence of less than or equal to 75% of the humans havingantibodies that bind to the CTLA4 portion of the CTLA4-Ig molecule. Inone embodiment, the incidence is 40-75%. In one embodiment, theincidence is less than or equal to 40%. In one embodiment, the incidenceis measured in a cell-based luciferase reporter assay.

The invention provides for a method for producing CTLA4-Ig protein, themethod comprising: (a) expanding mammalian cells that produce CTLA4-Igprotein, wherein the expanding is from a seed culture to a liquidculture of at least 10,000 L until the CTLA4-Ig protein is produced at ayield of at least about 0.5 grams of CTLA4-Ig protein per liter ofliquid culture, as determined by assessing an aliquot of the liquidculture; and (b) isolating the CTLA4-Ig protein from the at least 10,000L liquid culture, wherein the isolating occurs when the liquid cultureexhibits greater than or equal to about 6.0 moles of NANA per mole ofCTLA4-Ig dimer or to CTLA4-Ig molecule, as determined by assessing analiquot of the liquid culture. The method also provides for a method forproducing CTLA4-Ig protein, the method comprising: (a) expandingmammalian cells that produce CTLA4-Ig protein, wherein the expanding isfrom a seed culture to a liquid culture of at least 10,000 L until theCTLA4-Ig protein is produced at a yield of at least about 0.5 grams ofCTLA4-Ig protein per liter of liquid culture, as determined by assessingan aliquot of the liquid culture; and (b) isolating the CTLA4-Ig proteinfrom the at least 10,000 L liquid culture, wherein the isolating occurswhen the liquid culture exhibits from about 5.2 to about 7.6 moles ofsialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule, asdetermined by assessing an aliquot of the liquid culture. The methodalso provides for a method for producing CTLA4-Ig protein, the methodcomprising: (a) expanding mammalian cells that produce CTLA4-Ig protein,wherein the expanding is from a seed culture to a liquid culture of atleast 10,000 L until the CTLA4-Ig protein is produced at a yield of atleast about 0.5 grams of CTLA4-Ig protein per liter of liquid culture,as determined by assessing an aliquot of the liquid culture; and (b)isolating the CTLA4-Ig protein from the at least 10,000 L liquidculture, wherein the isolating occurs when the liquid culture has a celldensity of from about 33×105 viable cells per mL of liquid culture toabout 79×105 cells per mL of liquid culture. The invention also providesa method for producing CTLA4-Ig protein, the method comprising: (a)expanding mammalian cells that produce CTLA4-Ig protein, wherein theexpanding is from a seed culture to a liquid culture of at least 10,000L until the CTLA4-Ig protein is produced at a yield of at least about0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determinedby assessing an aliquot of the liquid culture; and (b) isolating theCTLA4-Ig protein from the at least 10,000 L liquid culture, wherein theisolating occurs when cell viability in the liquid culture is not lessthan about 38%. The invention also provides for a method for producingCTLA4-Ig protein, the method comprising: (a) expanding mammalian cellsthat produce CTLA4-Ig protein, wherein the expanding is from a seedculture to a liquid culture of at least 10,000 L until the CTLA4-Igprotein is produced at a yield of at least about 0.5 grams of CTLA4-Igprotein per liter of liquid culture, as determined by assessing analiquot of the liquid culture; and (b) isolating the CTLA4-Ig proteinfrom the at least 10,000 L liquid culture, wherein the isolating occurswhen cell viability in the liquid culture is not less than about 37%.The method also provides for a method for producing CTLA4-Ig protein,the method comprising: (a) expanding mammalian cells that produceCTLA4-Ig protein, wherein the expanding is from a seed culture to aliquid culture of at least 10,000 L until the CTLA4-Ig protein isproduced at a yield of at least about 0.5 grams of CTLA4-Ig protein perliter of liquid culture, as determined by assessing an aliquot of theliquid culture; and (b) isolating the CTLA4-Ig protein from the at least10,000 L liquid culture, wherein the isolating occurs when endotoxin isless than or equal to about 76.8 EU per mL of liquid culture, asdetermined by assessing an aliquot of the liquid culture. The methodalso provides for a method for producing CTLA4-Ig protein, the methodcomprising: (a) expanding mammalian cells that produce CTLA4-Ig protein,wherein the expanding is from a seed culture to a liquid culture of atleast 10,000 L until the CTLA4-Ig protein is produced at a yield of atleast about 0.5 grams of CTLA4-Ig protein per liter of liquid culture,as determined by assessing an aliquot of the liquid culture; and (b)isolating the CTLA4-Ig protein from the at least 10,000 L liquidculture, wherein the isolating occurs when endotoxin is less than orequal to about 4.8 EU per mL of liquid culture, as determined byassessing an aliquot of the liquid culture. The invention also providesfor a method for producing CTLA4-Ig protein, the method comprising: (a)expanding mammalian cells that produce CTLA4-Ig protein, wherein theexpanding is from a seed culture to a liquid culture of at least 10,000L until the CTLA4-Ig protein is produced at a yield of at least about0.5 grams of CTLA4-Ig protein per liter of liquid culture, as determinedby assessing an aliquot of the liquid culture; and (b) isolating theCTLA4-Ig protein from the at least 10,000 L liquid culture, wherein theisolating occurs only when bioburden is less than 1 colony forming unitper mL of liquid culture, as determined by assessing an aliquot of theliquid culture. The invention also provides for a method for producingCTLA4-Ig protein, the method comprising: (a) expanding mammalian cellsthat produce CTLA4-Ig protein, wherein the expanding is from a seedculture to a liquid culture of at least 10,000 L until the CTLA4-Igprotein is produced at a yield of at least about 0.5 grams of CTLA4-Igprotein per liter of liquid culture, as determined by assessing analiquot of the liquid culture; and (b) isolating the CTLA4-Ig proteinfrom the at least 10,000 L liquid culture, wherein the isolating occurswhen at least two of the following conditions are met: (i) the liquidculture contains greater than or equal to about 6.0 moles of NANA permole of CTLA4-Ig dimer or to CTLA4-Ig molecule; (ii) the liquid culturehas a cell density of from about 33×105 viable cells per mL of liquidculture to about 79×105 viable cells per mL of liquid culture; (iii) thecell viability in the liquid culture is not less than about 38%; or (iv)the yield of CTLA4-Ig protein is greater than about 0.5 grams ofCTLA4-Ig protein per liter of liquid culture, wherein NANA concentrationin (i) and yield in (iv) are determined by assessing an aliquot of theliquid culture. The invention also provides for a method for producingCTLA4-Ig protein, the method comprising: (a) expanding mammalian cellsthat produce CTLA4-Ig protein, wherein the expanding is from a seedculture to a liquid culture of at least 10,000 L until the CTLA4-Igprotein is produced at a yield of at least about 0.5 grams of CTLA4-Igprotein per liter of liquid culture, as determined by assessing analiquot of the liquid culture; and (b) isolating the CTLA4-Ig proteinfrom the at least 10,000 L liquid culture, wherein the isolating occurswhen at least two of the following conditions are met: (i) the liquidculture contains from about 5.2 to about 7.6 moles of sialic acid permole of CTLA4-Ig dimer or to CTLA4-Ig molecule; (ii) the cell viabilityin the liquid culture is not less than about 37%; or (iii) the yield ofCTLA4-Ig protein is greater than about 0.5 grams of CTLA4-Ig protein perliter of liquid culture, wherein the sialic acid content in (i) andyield in (iii) are determined by assessing an aliquot of the liquidculture.

Sequences:

[CTLA4-Ig nucleotide sequence, See FIG. 1] SEQ ID NO: 1 [CTLA4-Ig aminoacid sequence, See FIG. 1] SEQ ID NO: 2 [CTLA4^(A29YL104E)-Ig nucleotidesequence comprises nucleotides 79 to 1149 of the nucleic acid sequenceshown in FIG. 2] SEQ ID NO: 3 SEQ ID NO: 23 is the full nucleotidesequence shown in FIG. 2. This nucleotide sequence includes the codingsequence for the prosequence. [CTLA4^(A29YL104E)-Ig amino acid sequence,FIG. 3, without the pro-sequence] SEQ ID NO: 4 [amino acids 25-383 ofSEQ ID NO: 2] SEQ ID NO: 5MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK [aminoacids 26-383 of SEQ ID NO: 2] SEQ ID NO: 6AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK [amino acids27-383 of SEQ ID NO: 2] SEQ ID NO: 7MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK [amino acids25-382 of SEQ ID NO: 2] SEQ ID NO: 8MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG [amino acids26-382 of SEQ ID NO: 2] SEQ ID NO: 9AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG [amino acids27-382 of SEQ ID NO: 2] SEQ ID NO: 10MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG [amino acids25-383 of SEQ ID NO: 4] SEQ ID NO: 11MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK [aminoacids 26-383 of SEQ ID NO: 4] SEQ ID NO: 12AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK [amino acids27-383 of SEQ ID NO: 4] SEQ ID NO: 13MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK [amino acids25-382 of SEQ ID NO: 4] SEQ ID NO: 14MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG [amino acids26-382 of SEQ ID NO: 4] SEQ ID NO: 15AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG [amino acids27-382 of SEQ ID NO: 4] SEQ ID NO: 16MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG SEQ ID NO: 17[CTLA4 extracellular domain sequence] SEQ ID NO: 18MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD SEQ ID NO: 195′-AGAAAAGGGGCTGGAGAGATGGCTCAGTGGTTAAGAGCA-3′ SEQ ID NOS: 20-22 SEQ IDNO: 20 5′-GTACTCAGG SEQ ID NO: 21 AGTCAGAGAC SEQ ID NO: 22CGGCAGATCTCTGTGAGTTTGAGGCCAGCCTGGTCTACAAAGCAAGTT- 3′

CTLA4-Ig Monomers and Multimers

In certain embodiments, the invention provides cell lines having anexpression cassette that comprises SEQ ID NO:1 (FIG. 1A). Such anexpression cassette when expressed in mammalian cells, including CHOcells, can result in the production of N-and C-terminal variants, suchthat the proteins produced from the expression cassette can have theamino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ IDNO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ IDNO:2 (FIG. 1A). These proteins can be referred to herein as “SEQ ID NO:2monomers,” or monomers “having a SEQ ID NO:2 sequence.” These SEQ IDNO:2 monomers can dimerize, such that dimer combinations can include,for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i)and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv);(ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and(v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and(v); (v) and (vi); and, (vi) and (vi). These different dimercombinations can also associate with each other to form tetramerCTLA4-Ig molecules. These monomers, dimers, teramers, and othermultimers can be referred to herein as “SEQ ID NO:2 proteins” orproteins “having a SEQ ID NO:2 sequence.” While the cell lines canproduce these variants immediately upon translation, the variants canmore typically be a product of post-translational actions in the cells.The cell line also secretes CTLA4-Ig molecules. Abatacept refers to SEQID NO:2 proteins.

CTLA4-Ig molecules can include, for example, CTLA4-Ig proteins inmonomer, dimer, trimer, tetramer, pentamer, hexamer, or other multimericforms. CTLA4-Ig molecules can comprise a protein fusion with at least anextracellular domain of CTLA4 and an immunoglobulin constant region.CTLA4-Ig molecules can have wild-type or mutant sequences, for example,with respect to the CTLA4 extracellular domain and immunoglobulinconstant region sequences. CTLA4-Ig monomers, alone, or in dimer,tetramer or other multimer form, can be glycosylated.

In some embodiments, the invention provides populations of CTLA4-Igmolecules that have at least a certain percentage of dimer or othermultimer molecules. For example, the invention provides CTLA4-Igmolecule populations that are greater than 90%, 95%, 96%, 97%, 98%, 99%,or 99.5% CTLA4-Ig dimers. In one embodiment, the invention provides aCTLA4-Ig molecule population that comprises from about 95% to about99.5% CTLA4-Ig dimer and from about 0.5% to about 5% of CTLA4-Igtetramer. In another embodiment, the CTLA4-Ig molecule populationcomprises about 98% CTLA4-Ig dimer, about 1.5% CTLA4-Ig tetramer andabout 0.5% CTLA4-Ig monomer.

In one embodiment, the invention provides a population of CTLA4-Igmolecules wherein the population is substantially free of CTLA4-Igmonomer molecules. Substantially free of CTLA4-Ig monomer molecules canrefer to a population of CTLA4-Ig molecules that have less than 1%,0.5%, or 0.1% of monomers.

In one embodiment, the invention provides a population of CTLA4-Igmolecules wherein the population is substantially free of CTLA4-Igmultimers that are larger than dimers, such as tetramers, hexamers, etc.(e.g., high molecular weight species). Substantially free of CTLA4-Igmultimer molecules larger than dimers can refer to a population ofCTLA4-Ig molecules that have less than 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or0.1% of CTLA4-Ig multimers (e.g., high molecular weight species) largerthan dimeric form.

A CTLA4-Ig monomer molecule can have, for example, the amino acidsequence of: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2 (iii)27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ ID NO:2, or optionally (v)25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2. When an expressioncassette comprising the nucleic acid sequence of SEQ ID NO:1 isexpressed in CHO cells, the predominant monomer form expressed has theN-terminus amino acid residue of methionine (residue 27 of SEQ ID NO:2),which corresponds to the N-terminus amino acid residue of wild-typehuman CTLA4. However, because SEQ ID NO:1 also includes the codingsequence for an Oncostatin M Signal Sequence (nucleotides 11-88 of SEQID NO:1), the expressed protein from SEQ ID NO:1 contains an OncostatinM Signal Sequence. The signal sequence is cleaved from the expressedprotein during the process of protein export from the cytoplasm, orsecretion out of the cell. But cleavage can result in N-terminalvariants, such as cleavage between amino acid residues 25 and 26 of SEQID NO. 2 (resulting in an N-terminus of residue 26, i.e., the “Alavariant”), or between amino acid residues 24 and 25 of SEQ ID NO. 2(resulting in an N-terminus of residue 25, i.e., the “Met-Ala variant”),as opposed to cleavage between amino acid residues 26 and 27 of SEQ IDNO. 2 (resulting in an N-terminus of residue 27). For example, theMet-Ala variant can be present in a mixture of CTLA4-Ig molecules atabout 1%, and the Ala variant can be present in a mixture of CTLA4-Igmolecules at about 8-10%. In addition, the expressed protein from SEQ IDNO:1 can have C-terminus variants due to incomplete processing. Thepredominant C-terminus is the glycine at residue 382 of SEQ ID NO:2. Ina mixture of CTLA4-Ig molecules, monomers having lysine at theC-terminus (residue 383 of SEQ ID NO:2) can be present, for example, atabout 4-5%.

In one embodiment, a CTLA4-Ig molecule has the amino acid sequence ofSEQ ID NO: 5 as follows (which is the same as amino acids 25-383 of SEQID NO:2):

[SEQ ID NO: 5] MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK;

In another embodiment, a CTLA4-Ig molecule has the amino acid sequenceof

SEQ ID NO: 6 as follows (which is the same as amino acids 26-383 of SEQID NO:2):

[SEQ ID NO: 6] AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK;

In another embodiment, a CTLA4-Ig molecule has the amino acid sequenceof SEQ ID NO: 7 as follows (which is the same as amino acids 27-383 ofSEQ ID NO:2):

[SEQ ID NO: 7] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK;

In another embodiment, a CTLA4-Ig molecule has the amino acid sequenceof SEQ ID NO: 8 as follows (which is the same as amino acids 25-382 ofSEQ ID NO:2):

[SEQ ID NO: 8] MAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG;

In one embodiment, a CTLA4-Ig molecule has the amino acid sequence ofSEQ ID NO: 9 as follows (which is the same as amino acids 26-382 of SEQID NO:2):

[SEQ ID NO: 9] AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG;

In one embodiment, a CTLA4-Ig molecule has the amino acid sequence ofSEQ ID NO: 10 as follows (which is the same as amino acids 27-382 of SEQID NO:2):

[SEQ ID NO: 10] MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG.

A CTLA4-Ig monomer molecule can comprise an extracellular domain ofhuman CTLA4. In one embodiment, the extracellular domain can comprisethe nucleotide sequence of nucleotides 89-463 of SEQ ID NO:1 that codefor amino acids 27-151 of SEQ ID NO:2. In another embodiment, theextracellular domain can comprise mutant sequences of human CTLA4. Inanother embodiment, the extracellular domain can comprise nucleotidechanges to nucleotides 89-463 of SEQ ID NO:1 such that conservativeamino acid changes are made. In another embodiment, the extracellulardomain can comprise a nucleotide sequence that is at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides 89-463 ofSEQ ID NO:1.

A CTLA4-Ig monomer molecule can comprise a constant region of a humanimmunoglobulin. This constant region can be a portion of a constantregion; this constant region can have a wild-type or mutant sequence.The constant region can be from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgD or IgE. The constant region can be from a light chain or aheavy chain of an immunoglobulin. Where the constant region is from anIgG, IgD, or IgA molecule, the constant region can comprise one or moreof the following constant region domains: C_(L), CH1, hinge, CH2, orCH3. Where the constant region is from IgM or IgE, the constant regioncan comprise one or more of the following constant region domains:C_(L), C_(H)1, C_(H)2, C_(H)3, or C_(H)4. In one embodiment, theconstant region can comprise on or more constant region domains fromIgG, IgD, IgA, IgM or IgE.

In one embodiment, CTLA4-Ig dimers are comprised of two monomers,wherein each monomer can have the same or different amino acid sequence,and where the sequence can be the amino acid sequence of: (i) 26-383 ofSEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2,(iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383of SEQ ID NO:2. Such CTLA4-Ig monomers can dimerize through theextracellular domain of the human CTLA4 sequence via a cysteine aminoacid residue at position 146 of SEQ ID NO:2.

A CTLA4-Ig molecule can multimerize through the interaction of IgM orIgA constant region domains with a J chain protein. IgM and IgA areusually produced as multimers in association with an additionalpolypeptide chain, the J chain. In pentameric IgM, the monomers arecrosslinked by disulfide bonds to each other in the CH3 domain and tothe J chain through the CH4 domain. IgM can also form hexamers that lacka J chain where multimerization is achieved through disulfide bonds toeach. In dimeric IgA, the monomers have disulfide bonds to the J chainvia their CH3 domain and not each other. Thus, in one embodiment, theinvention provides CTLA4-Ig multimers, including dimers, pentamers, andhexamers, wherein the Ig portion comprises an IgM constant region orportion thereof or an IgA constant region or portion thereof. SuchCTLA4-Ig multimers based on IgM or IgA can include the J chain.

In one embodiment, a CTLA4-Ig monomer molecule (CTLA4 GenBank AccessionNo. 113253) comprises a modified human IgG1 hinge region (nucleotides464-508 of SEQ ID NO:1; amino acids 152-166 of SEQ ID NO:2) wherein theserines at amino acid residues 156, 162, and 165 of SEQ ID NO:2 havebeen engineered from cysteines present in the wild-type sequence.

In one embodiment, a CTLA4-Ig monomer molecule comprises a modifiedhuman IgGl CH2 region and a wild-type CH3 region (the modified humanIgGl C_(H)2 domain having nucleotides 509-838 of SEQ ID NO:1 and aminoacids 167-276 of SEQ ID NO:2; the human IgGl C_(H)3 domain havingnucleotides 839-1159 of SEQ ID NO:1 and amino acids 277-383 of SEQ IDNO:2).

In one embodiment, a CTLA4-Ig molecule population comprises monomershaving a sequence shown in any one or more of FIG. 7, 8, or 9 of theU.S. patent application published as Publication No. US 2002/0182211 A1,and in U.S. patent applications published as Publication Nos.US20030083246 and US20040022787, each of which is hereby incorporated byreference in its entirety.

In one embodiment, a CTLA4-Ig tetramer molecule comprises two pairs ortwo dimers of CTLA4-Ig polypeptides, wherein each polypeptide has one ofthe following amino acid sequences: (i) 26-383 of SEQ ID NO:2, (ii)26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. Eachmember of the pair of polypeptides or dimer is covalently linked to theother member, and the two pairs of polypeptides are non-covalentlyassociated with one another thereby forming a tetramer. Such tetramermolecules are capable of binding to CD80 or CD86. In another embodiment,such tetramer molecules can bind to CD80 or CD86 with an avidity that isat least 2-fold greater than the binding avidity of a CTLA4-Ig dimer(whose monomers have one of the above amino acid sequences) to CD80 orCD86. In another embodiment, such tetramer molecules can bind to CD80 orCD86 with an avidity that is at least 2-fold greater than the bindingaffinity or avidity of wild-type CTLA4 to CD80 or CD86. Such greateravidity can contribute to higher efficacy in treating immune disordersand other diseases as described below, as well as in inhibiting tissueand/or solid organ transplant rejections. In addition, greater orimproved avidity can produce the result of higher potency of a drug. Forexample, a therapeutic composition comprising CTLA4-Ig tetramer wouldhave a higher avidity and therefore higher potency than the same amountof a therapeutic composition having CTLA4-Ig monomer. In anotherembodiment, such tetramer molecules can have at least a 2-fold greaterinhibition on T cell proliferation as compared to a CTLA4-Ig dimer(whose monomers have one of the above amino acid sequences). In anotherembodiment, such tetramer molecules can have at least a 2-fold greaterinhibition on T cell proliferation as compared to a wild-type CTLA4molecule.

CTLA4^(A29YL104E)-Ig Monomers, Dimers, and Multimers

CTLA4^(A29YL104E)-Ig are modified forms of CTLA4-Ig (FIG. 1A; SEQ IDNOS: 1-2). The modification consists of point mutations that result intwo amino acid substitutions (L104E and A29Y) as shown in FIG. 2(corresponding to amino acid positions 55 and 130 in FIG. 3; SEQ ID NO:4). Relative to CTLA4-Ig, CTLA4^(A29YL104E)-Ig (for example, SEQ IDNOS:5-10) bind CD80 (B7-1) with approximately 2-fold increased avidity,and binds CD86 (B7-2) with approximately 4-fold increased avidity.CTLA4^(A29YL104E)-Ig are approximately 10-fold more effective thanCTLA4-Ig at inhibiting T cell proliferation, cytokine production, andCD28-dependent killing of target cells by natural killer cells.CTLA4^(A29YL104E)-Ig cause modest inhibition of B7-1 mediated T cellproliferation but are markedly more potent than CTLA4-Ig at blockingB7-2 mediated T cell proliferation. The increased potency is comparable,whether blocking B7-2 alone or blocking both B7-1 and B7-2, suggestingthat the enhanced immunomodulatory activity of CTLA4^(A29YL104E)-Ig canmost likely be attributed to the enhanced potency for blocking B7-2.

CTLA4^(A29YL104E)-Ig is a genetically engineered fusion protein, whichconsists of the functional binding domain of modified human CTLA-4 andthe Fc domain of human immunoglobulin of the IgGl class (FIG. 3A-3B).Two amino acid substitutions were made in the B7 binding region of theCTLA-4 domain (L104E and A29Y) to generate a CTLA4^(A29YL104E)-Igmolecule. A CTLA4^(A29YL104E)-Ig dimer is comprised of two glycosylatedCTLA4^(A29YL104E)-Ig chains. It exists as a covalent dimer linkedthrough an inter-chain disulfide bond. A molecular model of aCTLA4^(A29YL104E)-Ig molecule is shown in FIG. 4. A CTLA4^(A29YL104E)-Igmolecule has an average mass of approximately 91,800 Da as determined bymatrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF)mass spectrometry.

In certain embodiments, the invention provides cell lines having anexpression cassette that comprises SEQ ID NO:3. Such an expressioncassette when expressed in mammalian cells, for example CHO cells, canresult in the production of N-and C-terminal variants, such that thepolypeptides produced from the expression cassette can have the aminoacid sequence of residues: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQID NO:4; (iii) 27-383 of SEQ ID NO:4, or (iv) 27-382 of SEQ ID NO:4, oroptionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4.These polypeptides can be referred to herein as “SEQ ID NO:4 monomers,”or monomers “having a SEQ ID NO:4 sequence.”

These SEQ ID NO:4 monomers can dimerize, such that dimer combinationscan include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i)and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii)and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv);(iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and(vi); (v) and (v); (v) and (vi); and, (vi) and (vi). These differentdimer combinations can also associate with each other to form tetramerCTLA4^(A29YL104E)-Ig molecules. These monomers, dimers, teramers, andother multimers can be referred to herein as “SEQ ID NO:4 polypeptides”or polypeptides “having a SEQ ID NO:4 sequence.” While the cell linescan produce these variants immediately upon translation, the variantscan more typically be a product of post-translational actions in thecells. Dimers can be covalently joined together, non-covalently joinedtogether, or both. The invention provides for compositions that consistessentially of dimers that are covalently bound together. For example,the invention provides for compositions where at least 50% of theCTLA4-Ig dimers are made up of monomers joined covalently. The inventionalso provides for compositions where at least 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% of the CTLA4-Ig dimers are made up ofmonomers joined covalently. The invention also provides for compositionsof CTLA4-Ig molecules, wherin the molecules are predominantly in dimerform, and the dimers are predominantly formed by covalent linkages. Forexample, the invention provides for a composition wherein the majorityof the CTAL4-Ig dimers are joined covalently. It is possible that somefraction of the CTLA4-Ig dimers in the composition are joinednon-covalently.

CTLA4^(A29YL104E)-Ig molecules can include, for example,CTLA4^(A29YL104E)-Ig in monomer, dimer, trimer, tetramer, pentamer,hexamer, or other multimeric forms. CTLA4^(A29YL104E)-Ig molecules cancomprise a protein fusion with at least an extracellular domain ofmodified CTLA4 (SEQ ID NO:18) and an immunoglobulin constant region.CTLA4^(A29YL104E)-Ig molecules can have mutant sequences, for example,with respect to the modified CTLA4 extracellular domain andimmunoglobulin constant region sequences. CTLA4^(A29YL104E)-Ig monomers,alone, or in dimer, tetramer or other multimer form, can beglycosylated.

In some embodiments, the invention provides populations ofCTLA4^(A29YL104E)-Ig molecules that have at least a certain percentageof dimer or other multimer molecules. For example, the inventionprovides CTLA4^(A29YL104E)-Ig molecule populations that are greater than90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of CTLA4^(A29YL104E)-Ig dimers.In one embodiment, the invention provides a CTLA4^(A29YL104E)-Igmolecule population that comprises from about 95% to about 99.5% ofCTLA4^(A29YL104E)-Ig dimer and from about 0.5% to about 5% ofCTLA4^(A29YL104E)-Ig tetramer. In a further embodiment, the inventionprovides a CTLA4^(A29YL104E)-Ig molecule population that comprises fromabout 95% to about 99.5% of CTLA4^(A29YL104E)-Ig dimer, from about 0.5%to about 2.5% of CTLA4^(A29YL104E)-monomer, and from about 0.5% to about5% of CTLA4^(A29YL104E)-Ig tetramer. In another embodiment, theCTLA4^(A29YL104E)-Ig molecule population comprises about 96% ofCTLA4^(A29YL104E)-Ig dimer, about 2.5% of CTLA4^(A29YL104E)-Ig tetramer,and about 0.5% of CTLA4^(A29YL104E)-Ig monomer.

In one embodiment, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules wherein the population is substantiallyfree of CTLA4^(A29YL104E)-Ig monomers. Substantially free ofCTLA4^(A29YL104E)-Ig monomers can refer to a population ofCTLA4^(A29YL104E)-Ig molecules that have less than 1%, 0.5%, or 0.1% ofmonomers.

In another embodiment, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules wherein the population is substantiallyfree of CTLA4^(A29YL104E)-Ig multimers that are larger than dimers, suchas tetramers, hexamers, etc. Substantially free of CTLA4^(A29YL104E)-Igmultimers larger than dimers can refer to a population ofCTLA4^(A29YL104E)-Ig molecules that have less than 6%, 5%, 4%, 3%, 2%,1%, 0.5%, or 0.1% of CTLA4^(A29YL104E)-Ig multimers larger than dimers.

A CTLA4^(A29YL104E)-Ig monomer can have, for example, the amino acidsequence of: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4 (iii)27-383 of SEQ ID NO:4, or (iv) 27-382 of SEQ ID NO:4, or optionally (v)25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4. When an expressioncassette comprising the nucleic acid sequence of SEQ ID NO:3 or 23 isexpressed in CHO cells, the predominant monomer form expressed has theN-terminus amino acid residue of methionine (residue 27 of SEQ ID NO:4),which corresponds to the N-terminus amino acid residue of human CTLA4.However, because SEQ ID NO:23 also includes the coding sequence for anOncostatin M Signal Sequence (nucleotides 11-88 of SEQ ID NO:23), theexpressed protein from SEQ ID NO:23 contains an Oncostatin M SignalSequence.

The signal sequence is cleaved from the expressed protein during theprocess of protein export from the cytoplasm, or secretion out of thecell. But cleavage can result in N-terminal variants, such as cleavagebetween amino acid residues 25 and 26 of SEQ ID NO:4 (resulting in anN-terminus of residue 26, i.e., the “Ala variant”), or between aminoacid residues 24 and 25 SEQ ID NO:4 (resulting in an N-terminus ofresidue 25, i.e., the “Met-Ala variant”), as opposed to cleavage betweenamino acid residues 26 and 27 SEQ ID NO:4 (resulting in an N-terminusbeginning with the Met residue at amino acid position 27). For example,the Met-Ala variant can be present in a mixture of CTLA4^(A29YL104E)-Igmolecules at about 1%, and the Ala variant can be present in a mixtureof CTLA4^(A29YL104E)-Ig molecules at about 10-20%.

In addition, the expressed protein from a nucleic acid comprising SEQ IDNO:3 can have C-terminus variants due to incomplete processing. Thepredominant C-terminus is the glycine at residue 382 of SEQ ID NO:4. Ina mixture of CTLA4^(A29YL104E)-Ig molecules, monomers having lysine atthe C-terminus (residue 383 of SEQ ID NO:4) can be present, for example,at about 4-8%

In one embodiment, a CTLA4^(A29YL104E)-Ig molecule comprises the aminoacid sequence of SEQ ID NO: 11 as follows (which is the same as aminoacids 25-383 of SEQ ID NO:4):

[SEQ ID NO: 11] MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK.

In another embodiment, a CTLA4^(A29YL104E)-Ig molecule comprises theamino acid sequence of SEQ ID NO: 12 as follows (which is the same asamino acids 26-383 of SEQ ID NO:4):

[SEQ ID NO: 12] AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK.

In a further embodiment, a CTLA4^(A29YL104E)-Ig molecule comprises theamino acid sequence of SEQ ID NO: 13 as follows (which is the same asamino acids 27-383 of SEQ ID NO:4):

[SEQ ID NO: 13] MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.

In another embodiment, a CTLA4^(A29YL104E)-Ig molecule comprises theamino acid sequence of SEQ ID NO: 14 as follows (which is the same asamino acids 25-382 of SEQ ID NO:4):

[SEQ ID NO: 14] MAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG.

In one embodiment, a CTLA4^(A29YL104E)-Ig molecule has the amino acidsequence of SEQ ID NO: 15 as follows (which is the same as amino acids26-382 of SEQ ID NO:4):

[SEQ ID NO: 15] AMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG.

In a further embodiment, a CTLA4^(A29YL104E)-Ig molecule has the aminoacid sequence of SEQ ID NO: 16 as follows (which is the same as aminoacids 27-382 of SEQ ID NO:4):

[SEQ ID NO: 16] MHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYEGIGNGTQIYVIDPEPCPDSDQEPKSSDKTHTSPPSPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG.

A CTLA4^(A29YL104E)-Ig monomer comprises an extracellular domain ofhuman CTLA4, wherein two amino acid substitutions were made in theCTLA-4 domain (L104E and A29Y) (FIG. 5). In one embodiment, theextracellular domain can comprise the nucleotide sequence of nucleotides89-463 of SEQ ID NO:23 that code for amino acids 27-151 of SEQ ID NO:4.In another embodiment, the extracellular domain can comprise mutantsequences of human CTLA4 (such as single, double, and triple sitemutants in amino acids 27-151 of SEQ ID NO:4). In another embodiment,the extracellular domain can comprise nucleotide changes to nucleotides89-463 of SEQ ID NO:23 such that conservative amino acid changes aremade. In a further embodiment, the extracellular domain can comprisenucleotide changes to nucleotides 89-463 of SEQ ID NO:23 such thatnon-conservative amino acid changes are made. In another embodiment, theextracellular domain can comprise a nucleotide sequence that is at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to nucleotides89-463 of SEQ ID NO:23.

A CTLA4^(A29YL104E)-Ig monomer can comprise a constant region of a humanimmunoglobulin. This constant region can be a portion of a constantregion. This constant region also can have a wild-type or mutantsequence. The constant region can be from human IgG₁, IgG₂, IgG₃, IgG₄,IgM, IgA₁, IgA₂, IgD or IgE. The constant region can be from a lightchain or a heavy chain of an immunoglobulin. Where the constant regionis from an IgG, IgD, or IgA molecule, the constant region can compriseone or more of the following constant region domains: C_(L), C_(H)1,hinge, C_(H)2, or C_(H)3. Where the constant region is from IgM or IgE,the constant region can comprise one or more of the following constantregion domains: C_(L), C_(H)1, C_(H)2, C_(H)3, or C_(H)4. In oneembodiment, the constant region can comprise on or more constant regiondomains from IgG, IgD, IgA, IgM or IgE.

In one embodiment, CTLA4^(A29YL104E)-Ig dimers are comprised of twomonomers, wherein each monomer can have the same or different amino acidsequence, and where the sequence can be the amino acid sequence of: (i)26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi)25-383 of SEQ ID NO:4. Such CTLA4^(A29YL104E)-Ig monomers can dimerizethrough the extracellular domain of the human CTLA4 sequence via acysteine amino acid residue at position 146 of SEQ ID NO:4 (or cysteineamino acid residue at position 120 of FIG. 5).

A CTLA4^(A29YL104E)-Ig molecule can multimerize through the interactionof IgM or IgA constant region domains with a J chain protein. IgM andIgA are usually produced as multimers in association with an additionalpolypeptide chain, the J chain. In pentameric IgM, the monomers arecrosslinked by disulfide bonds to each other in the C_(H)3 domain and tothe J chain through the C_(H)4 domain. IgM can also form hexamers thatlack a J chain where multimerization is achieved through disulfide bondsto each. In dimeric IgA, the monomers have disulfide bonds to the Jchain via their C_(H)3 domain and not each other. Thus, in oneembodiment, the invention provides CTLA4^(A29YL104E)-Ig multimers,including dimers, pentamers, and hexamers, wherein the Ig portioncomprises an IgM constant region or portion thereof or an IgA constantregion or portion thereof. Such CTLA4^(A29YL104E)-Ig multimers based onIgM or IgA can include the J chain.

In one embodiment, a CTLA4^(A29YL104E)-Ig monomer comprises a modifiedhuman IgGl hinge region (nucleotides 464-508 of SEQ ID NO:23; aminoacids 152-166 of SEQ ID NO:4) wherein the serine residues at positions156, 162, and 165 of SEQ ID NO:4 have been engineered from cysteineresidues present in the wild-type sequence.

In one embodiment, a CTLA4^(A29YL104E)-Ig monomer comprises a modifiedhuman IgGl CH2 region and a wild-type CH3 region (the modified humanIgGl C_(H)2 domain having nucleotides 509-838 of SEQ ID NO:1 and aminoacids 167-276 of SEQ ID NO:2; the human IgGl C_(H)3 domain havingnucleotides 839-1159 of SEQ ID NO:1 and amino acids 277-383 of SEQ IDNO:2).

In one embodiment, a CTLA4^(A29YL104E)-Ig molecule population comprisesmonomers having a sequence shown U.S. Patent Application PublicationNos. U.S. 2002/0039577, U.S. 2003/0007968, U.S. 2004/0022787, U.S.2005/0019859 and U.S. 2005/0084933, and U.S. Pat. No. 7,094,874, each ofwhich is hereby incorporated by reference in its entirety.

In one embodiment, a CTLA4^(A29YL104E)-Ig tetramer comprises two pairsor two dimers of CTLA4^(A29YL104E)-Ig molecules, wherein eachpolypeptide has one of the following amino acid sequences: (i) 26-383 ofSEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4,(iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383of SEQ ID NO:4. Each member of the pair of polypeptides or dimer iscovalently linked to the other member, and the two pairs of polypeptidesare non-covalently associated with one another thereby forming atetramer. Such tetramer molecules are capable of binding to CD80 orCD86. In another embodiment, such tetramer molecules can bind to CD80 orCD86 with an avidity that is at least 2-fold greater than the bindingavidity of a CTLA4^(A29YL104E)-Ig dimer (whose monomers have one of theabove amino acid sequences) to CD80 or CD86.

Such greater avidity can contribute to higher efficacy in treatingimmune disorders and other diseases as described below, as well as ininhibiting tissue and/or solid organ transplant rejections. In addition,greater or improved avidity can produce the result of higher potency ofa drug. For example, a therapeutic composition comprisingCTLA4^(A29YL104E)-Ig tetramer would have a higher avidity and thereforehigher potency than the same amount of a therapeutic composition havingCTLA4^(A29YL104E)-Ig monomer. In another embodiment, such tetramermolecules can have at least a 2-fold greater inhibition on T cellproliferation as compared to a CTLA4^(A29YL104E)-Ig dimer (whosemonomers have one of the above amino acid sequences). In anotherembodiment, such tetramer molecules can have at least a 2-fold greaterinhibition on T cell proliferation as compared to a CTLA4-Ig tetramermolecule.

Characterization of CTLA4-Ig and CTLA4^(A29YL104E)-Ig Molecules

T cell proliferation can be measured using standard assays known in theart. For example, one of the most common ways to assess T cellproliferation is to stimulate T cells via antigen or agonisticantibodies to TCR and to measure, for example, the incorporation oftritiated thymidine (³H-TdR) in proliferating T cells or the amount ofcytokines released by proliferating T cells into culture. The inhibitoryeffect of CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules upon T cellactivation or proliferation can thereby be measured.

The affinity of a CTLA4-Ig molecule is the strength of binding of themolecule to a single ligand, including CD80, CD86, or CD80Ig or CD86Igfusion proteins. The affinity of CTLA4-Ig or CTLA4^(A29YL104E)-Ig toligands can be measured, for example, by using binding interactionanalysis (BIA) based on surface plasmon technique. Aside from measuringbinding strength, it permits real time determination of bindingkinetics, such as association and dissociation rate constants. A sensorchip, consisting of a glass slide coated with a thin metal film, towhich a surface matrix is covalently attached, is coated with one of theinteractants, Le, CTLA4-Ig, CTLA4^(A29YL104E)-Ig, or one of the ligands.A solution containing the other interactant is allowed to flow over itssurface. A continuous light beam is directed against the other side ofthe surface, and its reflection angle is measured. On binding ofCTLA4-Ig or CTLA4^(A29YL104E)-Ig to the ligand, the resonance angle ofthe light beam changes (as it depends on the refractive index of themedium close to the reactive side of the sensor, which in turn isdirectly correlated to the concentration of dissolved material in themedium). It is subsequently analyzed with the aid of a computer.

In one embodiment, CTLA4-Ig binding experiments can be performed bysurface plasmon resonance (SPR) on a BIAcore instrument (BIAcore AG,Uppsala, Sweden). CTLA4-Ig can be covalently coupled by primary aminegroups to a carboxymethylated dextran matrix on a BIAcore sensor chip,thereby immobilizing CTLA4-Ig to the sensor chip. Alternatively, ananti-constant region antibody can be used to immobilize CTLA4-Igindirectly to the sensor surface via the Ig fragment. Thereafter,ligands are added to the chip to measure CTLA4-Ig binding to theligands. Affinity measurements can be performed, for example, asdescribed in van der Merwe, P. et al., J. Exp. Med. (1997) 185(3):393-404, which is hereby incorporated by reference in its entirety.In another embodiment, CTLA4^(A29YL104E)-Ig binding experiments can beperformed using surface plasmon resonance (SPR) technology as describedabove (FIG. 6; see EXAMPLE 21).

The avidity of CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules can also bemeasured. Avidity can be defines as the sum total of the strength ofbinding of two molecules or cells to one another at multimple sites.Avidity is distinct from affininty, which is the strength of binding onesite on a molecule to its ligand. Without being bound by theory, higheravidity of CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules can lead toincreased potency of inhibiton by CTLA4-Ig or CTLA4^(A29YL104E)-Igmolecules on T-cell proliferation and activation. Avidity can bemeasured, for example, by two categories of solid phase assays: a)competitive inhibition assays, and b) elution assays. In both of themthe ligand is attached to a solid support. In the competitive inhibitionassay, CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules are then added insolution at a fixed concentration, together with free ligand indifferent concentrations, and the amount of ligand, which inhibits solidphase binding by 50%, is determined. The less ligand needed, thestronger the avidity. In elution assays, the ligand is added insolution. After obtaining a state of equilibrium, a chaotrope ordenaturant agent (e.g. isothiocyanate, urea, or diethylamine) is addedin different concentrations to disrupt CTLA4-Ig/ligand interactions orCTLA4^(A29YL104E)-Ig/ligand interactions. The amount of CTLA4-Ig orCTLA4^(A29YL104E)-Ig resisting elution is determined thereafter with anELISA. The higher the avidity, the more chaotropic agent is needed toelute a certain amount of CTLA4-Ig or CTLA4^(A29YL104E)-Ig. The relativeavidity of a heterogeneous mixture of CTLA4-Ig molecules orCTLA4^(A29YL104E)-Ig can be expressed as the avidity index (AI), equalto the concentration of eluting agent needed to elute 50% of the boundCTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules. Refined analysis of data canbe performed by determining percentages of eluted CTLA4-Ig orCTLA4^(A29YL104E)-Ig at different concentrations of the eluting agent.

A Phenyl Sepharose 4 Fast Flow column chromatography, HydrophobicInteraction Chromatography (HIC), process can be used to reduce theamount of CTLA4-Ig high molecular weight species eluted in a HICpurification step (see Example 15). Therefore, the cleaning peak fromthe HIC column is enriched in CTLA4-Ig HMW species. For example,preparative single or tandem column SEC HPLC can be employed to purifydimer, tetramer and multimer subpopulations from HIC cleaning peakmaterial. In one embodiment, the purified components are CTLA4-Ig dimer,tetramer, and hexamer. Characterization of high molecular weightcomponents of CTLA4-Ig present in the HIC cleaning peak can be done bystatic and dynamic light scattering techniques. Samples taken at thehydrophobic interaction chromatography (HIC) process step chase revealedthe presence of dimer, tetramer, and multimers at various samplingpoints. Hexamer species can be detected only in samples corresponding tothe “start of the cleaning peak” and “cleaning peak maximum OD”. Decamerspecies were detected in the “cleaning peak maximum OD” only. Molar massand hydrodynamic radius formation can be determined by fractionation viasize exclusion chromatography (SEC) employing MultiAngle lightscattering (MALS) coupled with quasi elastic light scatter (QELS)detection.

With respect to CTLA4-Ig molecules produced from the cell line, SECshows the Protein A eluate is a mixture of multimer, tetramer, and dimercomponents. Fractionation of this mixture on preparative tandem SECcolumn enables isolation of quantities of multimer, tetramer and dimerspecies. The area percent recovery for each component in SEC analysis ofthe isolated fractions results in 93-98% homogeneity for each fraction.In one aspect, purification of the individual components enablescomparison of the physicochemical properties of components of CTLA4-IgHMW material to those of CTLA4-Ig dimer. FIG. 7 shows the apparentmolecular weights, which correspond to multimer, tetramer, and dimerfractions of CTLA4-Ig HIC cleaning peak, as determined by SEC withdynamic light scattering detection (DSL) and retention time on SEC. Inone embodiment, the biospecific binding activity of purified componentsfrom the HIC cleaning peak is comparable to the binding activity ofdetermined by the BIAcore based immobilized B7-1Ig binding assay. Inanother aspect, sialic acid molar ratio for components isolated from HICcleaning peak are in the range of 4.9 to 7.6 whereas the sialic acidmolar ratio of CTLA4-Ig molecules or dimer (not in the HIC cleaningpeak) is in the range of 8-10. Analysis by IEF gel indicates reducedmobility CTLA4-Ig isoforms purified from HIC cleaning peak compared tothe migration of CTLA4-Ig dimer. This is consistent with lower sialicacid molar ratios observed for the CTLA4-Ig HIC cleaning peak fractions(FIG. 8).

The choice of cell culture conditions can influence the formation ofsingle chain (i.e., monomer) and high molecular weight species (i.e,dimers, tetramers, etc.) of a recombinant protein product. Growthconditions, also including but not limited to media composition, arefactors that can affect the formation of single chain, and the level ofcysteinylation. This is likely the result of presence of agents thatlead to disulfide bond reduction. The supplementation of cysteinedirectly or cysteine containing media to cells secreting CTLA4-Ig orCTLA4^(A29YL104E)-Ig can result in a rapid formation of single chain andhigh molecular weight species. The rate is proportional to amount ofcysteine added. In another embodiment, the supplementation ofiodoacetamide, a compound that reacts with free sulfhydryls, blocks theformation of high molecular weight species of CTLA4-Ig orCTLA4^(A29YL104E)-Ig that are dependent upon disulfide bonds.

For example, the iodoacetamide sensitive and non-sensitive highmolecular weight pathway highlight two major and distinctly differentmechanisms by which high molecular weight species can form in CTLA4-Ig.The supplementation of high salt concentrations (0.5M) to CTLA4-Igsolutions results in a sustained, rapid rate of high molecular weightformation. EDTA, ConAcidSol II, and yeastolates modestly increase singlechain formation (see Example 5).

In certain embodiments, the invention provides methods for generatinghigh molecular weight CTLA4-Ig populations, wherein mixtures containingpredominantly monomers or dimers of CTLA4-Ig are supplemented with highsalt such that the mixture has a salt concentration greater than about0.3, 0.4, 0.45, 0.5, or 0.6M. In one embodiment, such methods generate amixture comprising a CTLA4-Ig population that has at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CTLA4-Ig tetramer molecules.

In one embodiment, the invention provides a population of CTLA4-Igsingle chain species containing a modification on Cys¹⁴⁶ such that it iscysteinylated (see Example 4). Cysteinlyation is a posttranslationalmodification wherein a cysteine within a polypeptide chain is modifiedby the attachment of another cysteine via a disulfide bond.Cysteinylation of proteins have been implicated in modifying proteinbioactivity including immunogenicity and antigenicity of MHC Class-Irestricted viral determinants. In one embodiment, the invention providesa composition that comprises at least 1, 5, 10, 15, 20, 25, 50, 75,90,or 95% of cysteinylated single chain CTLA4-Ig molecules. In anotherembodiment of the invention, a CTLA4-Ig population has no more thanabout 1% CTLA4-Ig monomer molecules, or in another embodiement, lessthan 0.6% CTLA4-Ig monomer.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofsialic acid to CTLA4-Ig molecules of from about 5 to about 18. In someembodiments the average molar ratio of sialic acid to CTLA4-Ig moleculesis between from about X to about Y, inclusive of X and Y, where X isabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, and Y isabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18. In otherembodiments the average molar ratio of sialic acid to CTLA4-Ig moleculesis between from about X to about Y, inclusive of X and Y, where X isabout 4.0, 4.5, 5.0, 5.5 or 6.0, and Y is about 8.0, 8.5, 9.0, 9.5, or10.0. In other embodiments the average molar ratio of sialic acid toCTLA4-Ig molecules is between from about X to about Y, inclusive of Xand Y, where X is about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0 and Y isabout 11.0, 11.5, 12.0, 12.5 or 13.0. In other embodiments the averagemolar ratio of sialic acid to CTLA4-Ig molecules is from about 6 toabout 14, from about 7 to about 13, from about 8 to about 12, or fromabout 9 to about 11. In other embodiments the average molar ratio ofsialic acid to CTLA4-Ig molecules is from about 5 to about 9, from about5.5 to about 9.5, from about 6 to about 9, from about 6 to about 10, orfrom about 7 to about 10. In other embodiments the average molar ratioof sialic acid to CTLA4-Ig molecules is greater than or equal to 5, orgreater than or equal to 8. In certain embodiments, the sialic acid isN-acetyl neuraminic acid (NANA).

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofN-glycolyl neuraminic acid (NGNA) to CTLA4-Ig molecules of less than orequal to 2.5, less than or equal to 2.0, less than or equal to 1.5, lessthan or equal to 1.0, or less than or equal to 0.5.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules are greater than or equal to93.0 area percent, greater than or equal to 93.5 area percent, greaterthan or equal to 94.0 area percent, greater than or equal to 94.5 areapercent, greater than or equal to 95.0 area percent, greater than orequal to 95.5 area percent, greater than or equal to 96.0 area percent,greater than or equal to 96.5 area percent, or greater than or equal to97.0 area percent CTLA4-Ig dimers as determined by size exclusionchromatography and spectrophotometric detection. In some embodiments,the composition comprises CTLA4-Ig molecules, wherein the CTLA4-Igmolecules are greater than or equal to 95.0 area percent CTLA4-Igdimers, and less than or equal to 4.0 area percent high molecular weightspecies as determined by size exclusion chromatography andspectrophotometric detection. In some embodiments, the compositioncomprises CTLA4-Ig molecules, wherein the CTLA4-Ig molecules are greaterthan or equal to 95.0 area percent CTLA4-Ig dimers, and less than orequal to 5.0 area percent high molecular weight species as determined bysize exclusion chromatography and spectrophotometric detection.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules are of less than or equal to2.0 area percent, less than or equal to 1.5 area percent, less than orequal to 1.0 area percent, or less than or equal to 0.5 area percentarea percent CTLA4-Ig monomers (i.e., single chain) as determined bysize exclusion chromatography and spectrophotometric detection.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules are of less than or equal to5.0 area percent, less than or equal to 4.5 area percent, less than orequal to 4.0 area percent, less than or equal to 3.5 area percent, lessthan or equal to 3.0 area percent, less than or equal to 2.5 areapercent, less than or equal to 2.0 area percent, less than or equal to1.5 area percent, less than or equal to 1.0 area percent, or less thanor equal to 0.5 area percent CTLA4-Ig high molecular weight species(e.g., tetramer) as determined by size exclusion chromatography andspectrophotometric detection. In some embodiments, especially thoseinvolving concentrated compositions comprising CTLA4-Ig molecules, (suchas, for example, those for subcutaneous administration) the CTLA4-Igmolecules are of less than or equal to10 area percent, less than orequal to 9 area percent, less than or equal to 8 area percent, less thanor equal to 7 area percent, less than or equal to 6 area percentCTLA4-Ig high molecular weight species as determined by size exclusionchromatography and spectrophotometric detection.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises an amount of MCP-1 orMCP-1-like material less than or equal to 50 ppm, less than or equal to40 ppm, less than or equal to 38 ppm, less than or equal to 30 ppm lessthan or equal to 20 ppm, less than or equal to 10 ppm, 5 ppm, less thanor equal to 4 ppm, less than or equal to less than or equal to 3 ppm,less than or equal to 2 ppm or less than or equal to 1 ppm. The presentinvention provides a composition comprising CTLA4-Ig molecules, whereinthe composition comprises MCP-1 or MCP-1-like material at less than orequal to 50 ng/mg CTLA4-Ig molecules, less than or equal to 40 ng/mgCTLA4-Ig molecules, less than or equal to 38 ng/mg CTLA4-Ig molecules,less than or equal to 30 ng/mg CTLA4-Ig molecules, less than or equal to20 ng/mg CTLA4-Ig molecules, less than or equal to 10 ng/mg CTLA4-Igmolecules, less than or equal to 5 ng/mg, less than or equal to 4 ng/mgCTLA4-Ig molecules, less than or equal to 3 ng/mg CTLA4-Ig molecules,less than or equal to 2 ng/mg CTLA4-Ig molecules or less than or equalto 1 ng/mg CTLA4-Ig molecules. The present invention provides acomposition comprising CTLA4-Ig molecules and an amount of MCP-1(including the absence of MCP-1) wherein said composition is apharmaceutically acceptable composition.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofgalactose to CTLA4-Ig molecules of from about 6 to about 19. In someembodiments the average molar ratio of sialic acid to CTLA4-Ig moleculesis from about X to about Y, where X is about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17 or 18, and Y is about 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18 or 19. In other embodiments the average molar ratio ofgalactose to CTLA4-Ig molecules is between from about X to Y, inclusiveof X and Y, where X is about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or10.0 and Y is about 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, or16.0. In other embodiments the average molar ratio of galactose toCTLA4-Ig molecules is between from about X to about Y, inclusive of Xand Y, wherein X is about 6.0, 6.5, 7.0, 7.5 or 8.0 and Y is about 15.0,15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, or 19.0. In other embodimentsthe average molar ratio of galactose to CTLA4-Ig molecules is from aboutfrom about 7 to about 15, from about 8 to about 14, from about 9 toabout 13, from about 10 to about 12. In other embodiments the averagemolar ratio of galactose to CTLA4-Ig molecules is from about 7 to about18, from about 8 to about 17, from about 9 to about 17, from about 9 toabout 16, or from about 10 to about 15. In other embodiments the averagemolar ratio of galactose to CTLA4-Ig molecules is greater than or equalto 8.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio offucose to CTLA4-Ig molecules of from about 0.5 to about 12. In someembodiments the average molar ratio of sialic acid to CTLA4-Ig moleculesis between from about X to about Y, inclusive of X and Y, where X isabout 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or 4.5, and Y is about 7.0, 7.5,8.0, 8.5, 9.0, 9.5, 10.0 or 10.5. In other embodiments the average molarratio of fucose to CTLA4-Ig molecules is between from about X to Y,inclusive of X and Y, where X is about 2.9, 3.1, 3.3, 3.5, 3.7, 3.9, or4.1 and Y is about 7.9, 8.1, 8.3, 8.5, 8.7, 8.9, or 9.1. In otherembodiments the average molar ratio of fucose to CTLA4-Ig molecules isbetween from about X to Y, inclusive of X and Y, wherein X is about 1.0,1.5, 1.7, 1.9, 2.1, 2.3, or 2.5, and Y is about 8.7, 8.9, 9.1, 9.3, 9.6,9.9, 10.1, 10.3 or 10.5. In other embodiments the average molar ratio offucose to CTLA4-Ig molecules is from about from about 3.3 to about 8.5,from about 3.5 to about 8.3, from about 3.7 to about 8.1, from about 3.9to about 7.9. In other embodiments the average molar ratio of fucose toCTLA4-Ig molecules is from about 1.5 to about 9.5, from about 1.7 toabout 9.3, from about 1.9 to about 9.1, or from about 2.1 to about 8.9.In other embodiments the average molar ratio of fucose to CTLA4-Igmolecules is greater than or equal to 1.7.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofmannose to CTLA4-Ig molecules of from about 5 to about 25. In someembodiments the average molar ratio of sialic acid to CTLA4-Ig moleculesis between from about X to about Y, inclusive of X and Y, where X isabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21,and Y is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 or 24. In other embodiments the average molar ratio ofmannose to CTLA4-Ig molecules is between from about X to Y, inclusive ofX and Y, where Xis about 6.5, 7.0, 7.5, 7.7, 7.9, 8.1, 8.3, 8.5, 9.0,9.5, 10.0, 10.5, 11.0, 11.5 or 12.0 and Y is about 17.0, 17.5, 18.0,18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23, 23.5 or 24.0.In other embodiments the average molar ratio of mannose to CTLA4-Igmolecules is between from about X to Y, inclusive of X and Y, where X isabout 8, 8.5, 9.0, 9.5 10.0 or 11.0 and Y is about 17.0, 17.5, 18.0,18.5, 19.0, 19.5 or 20.0. In other embodiments the average molar ratioof mannose to CTLA4-Ig molecules is from about from about 6 to about 23,from about 7 to about 22, from about 7.7 to about 22, from about 8 toabout 21, from about 9 to about 20, from about 10 to about 19, fromabout 11 to about 19, and from about 11 to about 17. In otherembodiments the average molar ratio of mannose to CTLA4-Ig molecules isfrom about 8 to about 19, from about 9 to about 18, from about 10 toabout 17, or from about 11 to about 16. In other embodiments the averagemolar ratio of mannose to CTLA4-Ig molecules is greater than or equal to7.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules are of less than or equal to5.0 area percent, less than or equal to 4.5 area percent, less than orequal to 4.0 area percent, less than or equal to 3.5 area percent, lessthan or equal to 3.0 area percent, less than or equal to 2.5 areapercent, less than or equal to 2.0 area percent, less than or equal to1.5 area percent, less than or equal to 1.0 area percent, or less thanor equal to 0.5 area percent oxidized species. The present inventionprovides a composition comprising CTLA4-Ig molecules, wherein theCTLA4-Ig molecules are less than or equal to 5.0 area percent, less thanor equal to 4.5 area percent, less than or equal to 4.0 area percent,less than or equal to 3.5 area percent, less than or equal to 3.0 areapercent, less than or equal to 2.5 area percent, less than or equal to2.0 area percent, less than or equal to 1.5 area percent, less than orequal to 1.0 area percent, or less than or equal to 0.5 area percentdeamidated species. In some embodiments the composition comprisesCTLA4-Ig molecules, wherein the CTLA4-Ig molecules are less than orequal to 3.5 area percent oxidized species and less than or equal to 2.5area percent deamidated species.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises bacterial endotoxins LAL atless than or equal to 0.7 EU/mg CTLA4-Ig molecules, less than or equalto 0.6 EU/mg CTLA4-Ig molecules, less than or equal to 0.5 EU/mgCTLA4-Ig molecules, less than or equal to 0.42 EU/mg CTLA4-Ig molecules,less than or equal to 0.4 EU/mg CTLA4-Ig molecules, less than or equalto 0.35 EU/mg CTLA4-Ig molecules, less than or equal to 0.3 EU/mgCTLA4-Ig molecules, less than or equal to 0.25 EU/mg CTLA4-Ig molecules,less than or equal to 0.20 EU/mg CTLA4-Ig molecules, less than or equalto 0.15 EU/mg CTLA4-Ig molecules, or less than or equal to 0.05 EU/mgCTLA4-Ig molecules.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises bioburden at less than orequal to 2 CFU/10 mL, less than or equal to 1.5 CFU/10 mL, less than orequal to 1 CFU/10 mL, or less than or equal to 0.5 CFU/10 mL.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises DNA at less than or equalto 25 pg/mg CTLA4-Ig molecules, less than or equal to 20 pg/mg CTLA4-Igmolecules, less than or equal to 15 pg/mg CTLA4-Ig molecules, less thanor equal to 10 pg/mg CTLA4-Ig molecules, less than or equal to 5.0 pg/mgCTLA4-Ig molecules, less than or equal to 4.0 pg/mg CTLA4-Ig molecules,less than or equal to 3.5 pg/mg CTLA4-Ig molecules, less than or equalto 3.0 pg/mg CTLA4-Ig molecules, less than or equal to 2.5 pg/mgCTLA4-Ig molecules, less than or equal to 1.5 pg/mg CTLA4-Ig molecules,less than or equal to 1.0 pg/mg CTLA4-Ig molecules, or less than orequal to 0.5 pg/mg CTLA4-Ig molecules, or less than or equal to 0.20pg/ml CTLA4-Ig molecules.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises cellular protein (e.g., CHOprotein or CHOP) at less than or equal to 200 ng/mg CTLA4-Ig molecules,less than or equal to 150 ng/mg CTLA4-Ig molecules, less than or equalto 125 ng/mg CTLA4-Ig molecules, less than or equal to 100 ng/mgCTLA4-Ig molecules, less than or equal to 90 ng/mg CTLA4-Ig molecules,less than or equal to 80 ng/mg CTLA4-Ig molecules, 70 ng/mg CTLA4-Igmolecules, less than or equal to 60 ng/mg CTLA4-Ig molecules, less thanor equal to 50 ng/mg CTLA4-Ig molecules, less than or equal to 40 ng/mgCTLA4-Ig molecules, less than or equal to 30 ng/mg CTLA4-Ig molecules,less than or equal to 25 ng/mg CTLA4-Ig molecules, less than or equal to20 ng/mg CTLA4-Ig molecules, less than or equal to 15 ng/mg CTLA4-Igmolecules, less than or equal to 10 ng/mg CTLA4-Ig molecules, or lessthan or equal to 5 ng/mg CTLA4-Ig molecules. The present inventionprovides a composition comprising CTLA4-Ig molecules, wherein thecomposition comprises cellular protein at less than or equal to 200 ppm,less than or equal to 150 ppm, less than or equal to 125 ppm, less thanor equal to 100 ppm, less than or equal to 90 ppm, less than or equal to80 ppm, 70 ppm, less than or equal to 60 ppm, less than or equal to 50ppm, less than or equal to 40 ppm, less than or equal to 30 ppm, lessthan or equal to 25 ppm, less than or equal to 20 ppm, less than orequal to 15 ppm, less than or equal to 10 ppm, or less than or equal to5 ppm.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises Triton-X (e.g., TritonX-100) at less than or equal to 4.0 ng/mg CTLA4-Ig molecules, less thanor equal to 3.5 ng/mg CTLA4-Ig molecules, less than or equal to 3.0ng/mg CTLA4-Ig molecules, less than or equal to 2.5 ng/mg CTLA4-Igmolecules, less than or equal to 2.0 ng/mg CTLA4-Ig molecules, less thanor equal to 1.5 ng/mg CTLA4-Ig molecules, less than or equal to 1.0ng/mg CTLA4-Ig molecules, or less than or equal to 0.5 ng/mg CTLA4-Igmolecules. The present invention provides a composition comprisingCTLA4-Ig molecules, wherein the composition comprises Triton-X at lessthan or equal to 4.0 ppm, less than or equal to 3.5 ppm, less than orequal to 3.0 ppm, less than or equal to 2.5 ppm, less than or equal to2.0 ppm, less than or equal to 1.5 ppm, less than or equal to 1.0 ppm,or less than or equal to 0.5 ppm.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the composition comprises Protein A at less than orequal to 8.0 ng/mg CTLA4-Ig molecules, less than or equal to 7.5 ng/mgCTLA4-Ig molecules, less than or equal to 7.0 ng/mg CTLA4-Ig molecules,less than or equal to 6.5 ng/mg CTLA4-Ig molecules, less than or equalto 6.0 ng/mg CTLA4-Ig molecules, less than or equal to 5.5 ng/mgCTLA4-Ig molecules, less than or equal to 5.0 ng/mg CTLA4-Ig molecules,less than or equal to 4.5 ng/mg CTLA4-Ig molecules, less than or equalto 4.0 ng/mg CTLA4-Ig molecules, less than or equal to 3.5 ng/mgCTLA4-Ig molecules, less than or equal to 3.0 ng/mg CTLA4-Ig molecules,less than or equal to 2.5 ng/mg CTLA4-Ig molecules, less than or equalto 2.0 ng/mg CTLA4-Ig molecules, less than or equal to 1.5 ng/mgCTLA4-Ig molecules, less than or equal to 1.0 ng/mg CTLA4-Ig molecules,or less than or equal to 0.5 ng/mg CTLA4-Ig molecules. The presentinvention provides a composition comprising CTLA4-Ig molecules, whereinthe composition comprises Protein A at less than or equal to 8.0 ppm,less than or equal to 7.5 ppm, less than or equal to 7.0 ppm, less thanor equal to 6.5 ppm, less than or equal to 6.0 ppm, less than or equalto 5.5 ppm, less than or equal to 5.0 ppm, less than or equal to 4.5ppm, less than or equal to 4.0 ppm, less than or equal to 3.5 ppm, lessthan or equal to 3.0 ppm, less than or equal to 2.5 ppm, less than orequal to 2.0 ppm, less than or equal to 1.5 ppm, less than or equal to1.0 ppm, or less than or equal to 0.5 ppm.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofGlcNAc to CTLA4-Ig molecules of from about 10 to about 40. In someembodiments the average molar ratio of GlcNAc to CTLA4-Ig molecules isbetween from about X to about Y, inclusive of X and Y, where X is anywhole number between 10 and 39 and Y is any whole number between 11 and40. In other embodiments the average molar ratio of GlcNAc to CTLA4-Igmolecules is between from about X to Y, inclusive of X and Y, where X isabout 12, 14, 14, 15, 16 or 17, and Y is about 32, 33, 34, 35, 36 or 37.In other embodiments the average molar ratio of GlcNAc to CTLA4-Igmolecules is from about 12 to about 35, from about 13 to about 35, fromabout 14 to about 35, from about 15 to about 35.

The present invention provides a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules have an average molar ratio ofGalNAc to CTLA4-Ig molecules of from about 0.5 to about 7.0. In someembodiments the average molar ratio of GalNAc to CTLA4-Ig molecules isbetween from about X to about Y, inclusive of X and Y, where Xis 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or2.0, and Y is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 6.0, 7.0 or 8.0. Inother embodiments the average molar ratio of GalNAc to CTLA4-Igmolecules is between from about X to Y, inclusive of X and Y, where X isabout 0.6, 0.7, 0.8, 0.9, or 1.0, and Y is about 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1 or 4.2. In other embodiments the average molar ratioof GalNAc to CTLA4-Ig molecules is from about 0.7 to about 4.1, fromabout 0.8 to about 4.0, from about 0.9 to about 3.9, or about 1.0 toabout 3.8, or about 1.1 to about 3.7. In other embodiments the averagemolar ratio of GalNAc to CTLA4-Ig molecules is from about 1.6 to about3.7, from about 1.7 to about 3.6, from about 1.8 to about 3.5, or about1.9 to about 3.4.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig composition exhibits bands in pI ranges asdetermined on an isoelectric focusing gel (IEF gel) as follows: fromabout 10 to about 22 bands in the pI range of from about 4.3 to about5.6; cumulative bands intensity of from about 90% to about 110% in a pIrange from about 4.3 to about 5.3 and about 3 major bands in a pI rangefrom about 4.5 to about 5.2. In one embodiment, the bands in the rangeof from about 4.3 to about 5.6 is from about 5 to about 30, from about 6to about 29, from about 7 to about 28, from about 8 to about 27, fromabout 9 to about 26, from about 10 to about 25, from about 11 to about24, from about 12 to about 23, from about 13 to about 22, from about 14to about 21, from about 15 to about 20, from about 16 to about 19, fromabout 17 to about 20, from about 18 to about 19.

Glycosylated CTLA4-Ig and CTLA4^(A29YL104E)-Ig Molecules and PopulationsThereof

Without limitation, glycosylation can refer to the addition of complexoligosaccharide structures to a protein at specific sites within thepolypeptide chain. Glycosylation of proteins and the subsequentprocessing of the added carbohydrates can affect protein folding andstructure, protein stability, including protein half life, andfunctional properties of a protein. Protein glycosylation can be dividedinto two classes by virtue of the sequence context where themodification occurs, O-linked glycosylation and N-linked glycosylation.O-linked polysaccharides are linked to a hydroxyl group, usually to thehydroxyl group of either a serine or a threonine residue. O-glycans arenot added to every serine and threonine residue. O-linkedoligosaccharides are usually mono or biantennary, i.e. they comprise oneor at most two branches (antennas), and comprise from one to fourdifferent kinds of sugar residues, which are added one by one.

N-linked polysaccharides are attached to the amide nitrogen of anasparagine. Only asparagines that are part of one of two tripeptidesequences, either asparagine-X-serine or asparagine-X-threonine (where Xis any amino acid except proline), are targets for glycosylation.N-linked oligosaccharides can have from one to four branches referred toas mono-, bi-, tri-tetraantennary. The structures of and sugar residuesfound in N-and O-linked oligosaccharides are different. Despite thatdifference, the terminal residue on each branch of both N-and O-linkedpolysaccharide can be modified by a sialic acid molecule a modificationreferred as sialic acids capping. Sialic acid is a common name for afamily of unique nine-carbon monosaccharides, which can be linked toother oligosaccharides. Two family members are N-acetyl neuraminic acid,abbreviated as Neu5Ac or NANA, and N-glycolyl neuraminic acid,abbreviated as Neu5Gc or NGNA.

The most common form of sialic acid in humans is NANA.N-acetylneuraminic acid (NANA) is the primary sialic acid speciespresent in CTLA4-Ig molecules. However, it should be noted that minorbut detectable levels of N-glycolylneuraminic acid (NGNA) are alsopresent in CTLA4-Ig molecules. Furthermore, the method described hereincan be used to determine the number of moles of sialic acids for bothNANA and NGNA, and therefore levels of both NANA and NGNA are determinedand reported for CTLA4-Ig molecules. N-and 0-linked oligosaccharideshave different number of branches, which provide different number ofpositions to which sialic acid molecules can be attached. N-linkedologosaccharides can provide up to four attachment positions for sialicacids, while O-linked oligosaccharides can provide two sites for sialicacid attachment.

Glycosylated proteins (glycoproteins), many of which have been producedby recombinant DNA technology methods, are of great interest asdiagnostic and therapeutic agents. Many eukaryotic transmembraneproteins destined for the cell surface and secreted proteins arepost-translationally modified to incorporate N-linked and O-linkedcarbohydrate groups. N-linked oligosaccharides are attached toasparagine residues when they are part of the peptide motifAsn-X-Ser/Thr, where X can be any amino acid except proline. O-linkedoligosaccharides are attached to serine or threonine residues. Thestructures of N-linked and O-linked oligosaccharides as well as thesugar residues found in each can be different. One type of sugar that iscommonly found on both is N-acetylneuraminic acid (NANA; hereafterreferred to as sialic acid). Usually, sialic acid is the terminalresidue of both N-linked and O-linked oligosaccharides. Theglycoprotein, because of its negative charge, can exhibit acidicproperties.

Glycosylated proteins are purported to play roles in augmenting proteinfolding, regulating cell sorting and trafficking, preventing proteinaggregation, mediating cell-cell adhesion, and increasing resistance toproteolysis. In eukaryotic organisms, the nature and extent ofglycosylation can have a profound impact on the circulating half-lifeand bioactivity of glycoprotein therapeutics by processes which involvereceptor mediated endocytosis and clearance. Receptor-mediated systemsare thought to play a major role in clearing serum glycoproteins byrecognizing the various sugar components of the oligosaccharide. Aglycoprotein's terminal sialic acid group can affect absorption,half-life, and serum clearance. Thus, glycoprotein productionstrategies, which maintain the terminal sialic acid component of theglycosylated protein, can better increase the protein's bioavailabilityand serum half-life. Several production process parameters have beeninvestigated pertaining to recombinant glycoprotein synthesis,especially the effect of media composition and temperature shifts invarious production strategies.

CTLA4-Ig dimers composed of monomers having the amino acid sequence ofresidues (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii)27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ IDNO:2, or (vi) 25-383 of SEQ ID NO:2, can have a predicted theoretical MWof about 78,000 to about 79,000 Daltons. However, the MW for such dimersobtained by MALDI-TOF is approximately 91,000 Daltons. This differencein MW of approximately 13,000-14,000 Daltons is due at least in part toglycosylation, which in one embodiment, accounts for approximately 15%of the mass of this particular CTLA4-Ig monomer molecule. The abovespecified monomers have three N-linked glycosylation sites that havebeen confirmed by peptide mapping to occur at asparagines at residues102, 134, and 233 of SEQ ID NO:2. Carbohydrate molecules that are linkedthrough asparagine can be cleaved selectively using the enzyme Peptide-NGlycosidase F (PNGase F). In one instance, treatment of the monomerhaving the sequence 27-383 of SEQ ID NO:2 with PNGase F resulted in aspecies with a MW of approximately 80,200 Daltons, and because thetheoretical MW of this monomer is about 80,200, the treatment suggestedthat the unaccounted 1,400 Daltons (80,200-78,800=1,400) can be due toO-linked glycosylation. Although there are numerous serine and threonineresidues that have the potential of being glycosylation sites, only twoO-linked sites were identified: Ser¹⁵⁵ and Ser¹⁶⁵ of SEQ ID NO:2. In oneembodiment, the predominant glycan attached to these two sites isHexNAc-Hex-NeuAc.

For example, FIG. 9 presents an overall view of the N-linked andO-linked carbohydrate structures on CTLA4-Ig molecules comprised ofmonomers having a sequence from SEQ ID NO:2 (Le, a monomer having one ofthe following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2), (v)25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2 wherein in oneembodiment such molecules with the shown carbohydrate characteristicsare produced by the cell-line of the invention or progeny thereofaccording to the method of production described in Examples 14-15. Themajor structures listed for each site are based on the orthogonaltechniques (see herein). For each structure there is an estimatedpercentage of that structure observed during these experiments. Thesepercentages represent best estimates from the orthogonal techniques.

CTLA4^(A29YL104E)-Ig dimers composed of monomers having the amino acidsequence of residues (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ IDNO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v)25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ ID NO:4, can have apredicted theoretical MW of about 78,000 to about 79,000 Daltons.However, the MW for such dimers obtained by MALDI-TOF is approximately91,500 Daltons. This difference in MW of approximately 12,000-13,000Daltons is due at least in part to glycosylation. The above specifiedmonomers have three N-linked glycosylation sites that have beenconfirmed by peptide mapping to occur at asparagines at residues 102,134, and 233 of SEQ ID NO:4 (N76, N108, and N207 of FIG. 4).Carbohydrate molecules that are linked through asparagine can be cleavedselectively using the enzyme Peptide-N Glycosidase F (PNGase F).Although there are numerous serine and threonine residues that have thepotential of being glycosylation sites, only three O-linked sites wereidentified: Ser149, Ser155, and Ser165 of SEQ ID NO:4 (See Table 25 inEXAMPLE 22) In one embodiment, the predominant glycan attached to thesesites is HexNAc-Hex-NeuAc.

In certain embodiments, CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules areglycoproteins that can be produced by the culture methods of theinvention. In one embodiment, CTLA4-Ig glycoproteins are modified witholigosaccharides that represent approximately 15% (w/w) of the molecule.These oligosaccharides can play an important role in the pharmacokinetic(PK) parameters of a CTLA4-Ig or CTLA4^(A29YL104E)-Ig glycoprotein. Inaddition, different oligosaccharide profiles can influence the stabilityand degradation of proteins. For example, O-linked oligosaccharides mayenhance the stability of CTLA4^(A29YL104E)-Ig molecules by preventingautolysis in the hinge region of the immunoglobulin constant region.

The oligosaccharide distribution on a population of CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules can be heterogeneous in nature due to thecomplexity of cell culture and processes. The heterogeneity can bepresent due to glycosylation sites being completely occupied tounoccupied, and the fact that any specific site can be populated withmany different oligosaccharide structures, which can further displayvariation in the pattern of sialic acid modification.

In one embodiment, the primary sialic acid moiety on CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules is N-acetyl neuraminic acid (NeuAc,NANA), and the secondary sialic acid moiety is N-glycolyl neuraminicacid (NGNA). The charged nature of sialic acid and the complex sialicacid-containing structures can result in multiple isoforms of CTLA4-Igor CTLA4^(A29YL104E)-Ig, respectively, where such isoforms can beevident in an isoelectric focusing (IEF) profile. For example, see FIG.10 and Example 3 for IEF profile of CTLA4-Ig. Additionally, see FIG. 11and EXAMPLE 22 for IEF profile of CTLA4^(A29YL104E)-Ig.

In one embodiment, the invention provides a population of CTLA4-Igmolecules that have a dominant CTLA4-Ig isoform having an isoelectricpoint (pI) that is less than or equal to 5.1 or 5.0, which can bedetermined for example by IEF. In another embodiment, a population ofCTLA4^(A29YL104E)-Ig molecules is provided that has dominantCTLA4^(A29YL104E)-Ig isoforms having an isoelectric point (pI) that isless than or equal to 5.5, which can be determined for example by IEF(FIG. 11).

In one embodiment, the invention provides a population of CTLA4-Igmolecules that have a pI of from about 4.2 to about 5.7, from about 4.25to about 5.5, from about 4.3 to about 5.3, or from about 4.5 to about5.2. In another embodiment, the invention provides a population ofCTLA4-Ig molecules that have a pI of from about 4.45 to about 5.30. In afurther embodiment, the invention provides a population of CTLA4-Igmolecules that have a pI of from about 4.3 to about 5.1. In a particularembodiment, the invention provides a population of CTLA4-Ig moleculesthat have a pI of from about 4.45 to about 5.0. In one embodiment, theinvention provides a population of CTLA4-Ig molecules where at least40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the populationexhibit an isoelectric point less than or equal to about 5.7, 5.6, 5.5,5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4. 4.3, 4.2, 4.1,4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7,2.6, 2.5, 2.4, 2.3, 2.2, 2.1, or 2.1 as determined by IEF (these valuescan have a Standard Deviation of±0.2). In one embodiment, the inventionprovides a method for preparing a population of CTLA4-Ig moleculeshaving a pI of from about 4.45 to about 5.30, or from about 4.45 toabout 5.1, or from about 4.45 to about 5.0, wherein the methods involvessubjecting a population of CTLA4-Ig molecules to IEF gelelectrophoresis, wherein a single band on the gel represents asub-population of CTLA4-Ig molecules having a particular pI, andisolating the sub-population of CTLA4-Ig molecules having the particularpI by excising the band from the gel and subsequent purification of theproteins from the excised gel band.

In further embodiments, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules that have a pI of from about 4.5 to about5.2. In other embodiments, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules that have a pI of from about 4.7 to about5.1. In another embodiment, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules that have a pI of from about 2.0 to about5.2. In one embodiment, the invention provides a population ofCTLA4^(A29YL104E)-Ig molecules where at least 40%, 50%, 60%, 70%, 80%,90%, or 95% of the molecules in the population exhibit an isoelectricpoint less than or equal to about 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9,4.8, 4.7, 4.6, 4.5, 4.4. 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5,3.4, 3.3, 3.2, 3.1, or 3.0 as determined by IEF (these values can have aStandard Deviation of±0.2). In one embodiment, the invention provides amethod for preparing a population of CTLA4^(A29YL104E)-Ig moleculeshaving a pI of from about 4.5 to about 5.2; from about 4.7 to about 5.1;from about 2.0 to about 5.2, wherein the method involves subjecting apopulation of CTLA4^(A29YL104E)-Ig molecules to IEF gel electrophoresis,wherein a single band on the gel represents a sub-population ofCTLA4^(A29YL104E)-Ig molecules having a particular pI, and isolating thesub-population of CTLA4^(A29YL104E)-Ig molecules having the particularpI by excising the band from the gel and subsequent purification of theproteins from the excised gel band.

In certain embodiments, the invention provides populations of CTLA4-Igmolecules having an average molar ratio of moles sialic acid groups tomoles CTLA4-Ig molecules of from: about 6 to about 32, about 8 to about32, about 11 to about 30, about 12 to about 20, about 13 to about 19,about 14 to about 18, about 15 to about 17, about 6 to about 16, about 8to about 16, about 8 to about 14, about 8 to about 12.

In some embodiments, a maximum allowable CHO host cell protein of ppm to10 ng/mg characterizes the composition of CTLA4-Ig molecules. In anotherembodiment, the composition of CTLA4-Ig molecues is characterized byhost cell DNA at a level of 2.5 pg/mg to 1.0 pg/mg. In anotherembodiment, the composition of CTLA4-Ig molecues is characterized byTriton X-100 at a level of 1.0 ng/mg or 1.0 ppm. The concentration ofTriton X-100 can be determined by extraction of the Triton X-100 usingWaters OASIS-HLB solid-phase extraction followed by washing with waterto remove residual protein. The bound Triton X-100 is removed by elutionwith acetonitrile. The acetonitrile eluate is analyzed by reversed-phasechromatography using a SAS Hypersil 5 μm column and a mobile phaseconsisting of acetonitrile:water (80:20). Detection is by UV absorbanceat 225 nm. In one embodiment, the composition of CTLA4-Ig molecules ischaracterized by ≤2.5 area % oxidation and ≤2.0 area % deamidation. Inanother embodiment, the composition of CTLA4-Ig molecules ischaracterized by ≤3.0 area % oxidation and ≤2.5 area % deamidation. Thetryptic peptide mapping method was used for quantitation of oxidationand deamidation. The percent oxidation data was determined by the use ofan RP-HPLC tryptic mapping assay that quantitates the area percentoxidation of Met85 in the CTLA4-Ig protein to methionine sulfoxide.Percent oxidation in the method is obtained by measuring UV peak areasin the RP-HPLC tryptic map for the T6 tryptic peptide, comprised ofresidues 84-93 containing Met85, and the corresponding oxidized trypticpeptide, T6ox, containing Met(O)85. The area percent oxidation of Met85to Met(O)85 is proportional to the area percent of the T6ox peak:Percent Oxidation=100*9 At6ox/(AT6ox+AT6), where, AT6=peak area for T6tryptic peptide, (84-93). AT6ox=peak area for T6ox tryptic peptide,Met(O)85(84-93). The percent deamidation data, acquired by using aRP-HPLC tryptic mapping assay that quantitates the area percentoxidation and deamidation, is obtained by measuring UV peak areas in theRP-HPLC tryptic map for the T26 tryptic peptide, comprised of residues281-302 containing Asn294, and the corresponding deamidated trypticpeptide, T26deam1, containing isoAsp294. The area percent deamidation ofAsn294 to isoAsp294, then, is proportional to the area percent of theT26deam1 peak: where, AT26=peak area for T26, (281-302), AT26deam1=peakarea for T26deam1, isoAsp294(281-302). AT26deam2=peak area for T26deam1,Asp299(281-302). AT26deam3=peak area for T26deam3, Asp294(281-302).AT26deam4=peak area for T26deam4, Asu294(281-302).

In another embodiment, the composition of CTLA4-Ig molecules ischaracterized by N-Acetylglucosamine (GlcNAc) of from 15 to 35Moles:Mole CTLA4-Ig Protein, or N-Acetylgalactosamine (GalNAc) of from1.7 to 3.6 Moles:Mole CTLA4-Ig Protein. The amino monosaccharides arequantitated by capillary electrophoresis (CE) following release from theprotein by acid hydrolysis. The released amino monosaccharides arere-acetylated, and fluorescently labeled with aminopyrene trisulfonicacid (APTS) to facilitate their detection and quantitation.N-Acetylmannosamine is added to a sample and amino monosaccharidestandards to serve as an internal standard. The peak areas of the aminomonosaccharides in the samples are normalized using the internalstandard and quantified by comparing with their respective normalizedamino monosaccharides peak areas in the standard. The molar ratio ofeach monosaccharide relative to the CTLA4-Ig molecule is thencalculated.

In one embodiment, the composition of CTLA4-Ig molecues is characterizedby the following N-linked oligosaccharide profile specifications:

N-Linked Oligosaccharide Profile Specifications % Difference %Difference % Difference Domain I Domain II Domain III % Difference 19-317-19 −6-−18 Standard Deviation ±29 ±27 ±25 (% Difference from abovespecification)

In one embodiment, the composition of CTLA4-Ig molecules ischaracterized by neutral monosaccharide where the composition has ratiosof about:

Galactose: 8.0 to 17 Moles:Mole CTLA4-Ig Protein

Fucose: 3.5 to 8.3 Moles:Mole CTLA4-Ig Protein

Mannose: 7.7 to 22 Moles:Mole CTLA4-Ig Protein, or

Galactose: 9.0 to 17 Moles:Mole CTLA4-Ig Protein

Mannose: 11 to 19 Moles:Mole CTLA4-Ig Protein.

Illustrative Neutral Monosaccharide Composition: Moles:Mole Protein ofGalactose, Fucose and Mannose Neutral Mono- Process W (n = 34) ProcessCD-CHO1 (n = 109) saccharide Mean (SD) Min, Max Mean (SD) Min, MaxGalactose 13.9 (1.1) 12.0, 16.0 12.6 (1.0) 10.0, 16.0 Fucose  5.8 (1.0)4.2, 7.7  5.6 (0.7) 4.5, 7.6 Mannose 15.3 (1.0) 13.0, 17.0 15.4 (1.0)13.0, 18.0

Illustrative Sialic Acid (NANA:Mole Protein) Process W (n = 34) ProcessCD-CHO1 (n = 109) Sialic Acid Mean (SD) Min, Max Mean (SD) Min, Max NANA10.2 (0.6) 9.3, 11.6 9.7 (0.6) 8.2, 11.5

In another embodiment, the monosaccharide molar ratio range for aCTLA4^(A29YL104E)-Ig composition is as follows: mannose from about 10-20moles/mole protein; fucose from about 4.2 -7.0 moles/mole protein; andgalactose from about 9.2-17 moles/mole protein. In another embodiment,the CTLA4^(A29YL104E)-Ig composition is characterized by a NANA molarratio of from about 5.0-10.0 mole of NANA/mole protein. In anotherembodiment, the CTLA4^(A29YL104E)-Ig composition is characterized by aNGNA molar ratio of <1.5 mole NGNA/mole protein. In some embodiment, the% deviation of molar ratio for sialic acids is 15% or 20% or 30%.

In one embodiment, a population of CTLA4-Ig molecules can compriseCTLA4-Ig monomers that each have at least 3 sialic acid groups. Inanother embodiment a population of CTLA4-Ig molecules comprises CTLA4-Igmonomers that each have from 3 to 8 sialic acid groups.

In one embodiment, the invention provides a population of CTLA4-Igmolecules where at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% of themolecules in the population exhibit an isoelectric point less than orequal to about 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7,4.6, 4.5, 4.4. 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3,3.2, 3.1, or 3.0.

In some embodiments, the invention provides populations of CTLA4-Igmolecules having an average molar ratio of moles NANA to moles CTLA4-Igmolecules or dimer of from: about 6 to about 16, about 6 to about 14,about 6 to about 12, about 8 to about 12, about 8 to about 14, about 8to about 16.

In other embodiments, the invention provides populations of CTLA4-Igmolecules having an average molar ratio of moles NGNA to moles CTLA4-Igmolecules or dimer of less than or equal to about 2, 1.8, 1.6, 1.5, 1.4,1.0, 0.8, or 0.5

In particular embodiments, the invention provides populations ofCTLA4^(A29YL104E)-Ig molecules having an average molar ratio of molessialic acid groups to moles CTLA4^(A29YL104E)-Ig molecules or dimer offrom about 5.5 to about 8.5. In another embodiment, the inventionprovides populations of CTLA4^(A29YL104E)-Ig molecules having an averagemolar ratio of moles sialic acid groups to moles CTLA4^(A29YL104E)-Igmolecules or dimer of from about 5 to about 10.

In one embodiment, a population of CTLA4^(A29YL104E)-Ig molecules cancomprise CTLA4^(A29YL104E)-Ig monomers that each have at least 2.5sialic acid groups. In another embodiment a population ofCTLA4^(A29YL104E)-Ig molecules comprises CTLA4^(A29YL104E)-Ig monomersthat each have from 2.5 to 5 sialic acid groups.

In other embodiments, the invention provides populations of CTLA4-Igmolecules that are distinguished by the population's average molar ratioof moles amino monosaccharides and/or neutral monosaccharides and/orsialic acids to moles CTLA4-Ig molecules or dimer. In particularembodiments, the invention provides populations of CTLA4^(A29YL104E)-Igmolecules that are distinguished by the population's average molar ratioof moles amino monosaccharides and/or neutral monosaccharides and/orsialic acids to moles CTLA4^(A29YL104E)-Ig molecules or dimer. Aminomonosaccharides include N-acetyl galactosamine (GalNAc) and N-acetylglucosamine (GlcNAc). Neutral monosaccharides include mannose, fucose,and galactose. Sialic acids include N-acetyl neuraminic acid (NANA) andN-glycolyl neuramininc acid (NGNA).

In one embodiment, the invention provides a population of CTLA4-Igmolecules that are characterized by an average molar ratio of molesGlcNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is fromabout 10 to about 40, from about 15 to about 35, from about 15 to about25, or from about 15 to about 20. In another embodiment, the inventionprovides a population of CTLA4-Ig molecules where at least 40%, 50%,60%, 70%, 80%, 90%, or 95% of the molecules in the population arecharacterized by an average molar ratio of moles GlcNAc per mole ofCTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal toabout 40, 38, 35, 30, 25, 20, 18, or 15.

In another embodiment, the invention provides a population of CTLA4-Igmolecules that are characterized by an average molar ratio of molesGalNAc per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is fromabout 1.5 to about 8.5, from about 1.7 to about 3.0, from about 1.7 toabout 4.0, from about 1.7 to about 5.0, from about 1.7 to about 6.0,from about 1.7 to about 7.0, from about 1.7 to about 8.0, or from about1.7 to about 8.3. In another embodiment, the invention provides apopulation of CTLA4-Ig molecules where at least 40%, 50%, 60%, 70%, 80%,90%, or 95% of the molecules in the population are characterized by anaverage molar ratio of moles GalNAc per mole of CTLA4-Ig dimer or toCTLA4-Ig molecule that is less than or equal to about 8.5, 8, 7.5, 7,6.5, 6, 5.5, 5, 4.5, 4.0, 3.8, 3.6, 3.5, 3.0, 2.5, 2.0, 1.7, or 1.5.

In a further embodiment, the invention provides a population of CTLA4-Igmolecules that are characterized by an average molar ratio of molesgalactose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that isfrom about 7.5 to about 20.0, from about 8.0 to about 19.0, from about 8to about 18.0, from about 8.0 to about 17.0, from about 8.5 to about17.0, or from about 9.0 to about 17.0. In another embodiment, theinvention provides a population of CTLA4-Ig molecules where at least40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the populationcharacterized by an average molar ratio of moles galactose per mole ofCTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal toabout 20.0, 19.0, 18.0, 17.0, 16.0, 15.0, 14.0, 13.0, 12.0, 11.0, 10.0,9.0, 8.5, 8.0, or 7.5.

In a further embodiment, the invention provides a population of CTLA4-Igmolecules that are characterized by an average molar ratio of molesfucose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is fromabout 3 to about 8.5, from about 3.5 to about 8.5, from about 3.5 toabout 8.3, from about 3.5 to about 8.0, from about 3.5 to about 7.5, orfrom about 3.5 to about 7.0. In another embodiment, the inventionprovides a population of CTLA4-Ig molecules where at least 40%, 50%,60%, 70%, 80%, 90%, or 95% of the molecules in the populationcharacterized by an average molar ratio of moles fucose per mole ofCTLA4-Ig dimer or to CTLA4-Ig molecule that is less than or equal toabout 8.5, 8.3, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.2,or 3.0.

In a further embodiment, the invention provides a population of CTLA4-Igmolecules that are characterized by an average molar ratio of molesmannose per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule that is fromabout 7 to about 23, from about 7.5 to about 23, from about 7.7 to about23, from about 7.7 to about 22.5, from about 7.7 to about 22, from about7.7 to about 20, from about 7.7 to about 18, from about 7.7 to about 16,from about 8.0 to about 16.0, from about 9.0 to about 17.0, from about10 to about 19.0, or from about 11 to about 19.0. In another embodiment,the invention provides a population of CTLA4-Ig molecules where at least40%, 50%, 60%, 70%, 80%, 90%, or 95% of the molecules in the populationcharacterized by an average molar ratio of moles mannose per mole ofCTLA4-Ig molecules or dimer or to CTLA4-Ig molecule that is less than orequal to about 23, 22.5, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9.5, 9, 8.5, 8, 7.7, 7.5, 7.3, or 7.

In one embodiment, the invention provides a glycosylated CTLA4-Igpopulation that exhibits increased PK values, such as increased exposureas measured by area under the curve (AUC), such as resulting from or asdemonstrated by decreased clearance from the serum while retainingbioactivity. In another embodiment, the invention provides aglycosylated CTLA4^(A29YL104E)-Ig population that exhibits increasedpharmacokinetic (PK) values as demonstrated by decreased clearance fromthe serum while retaining bioactivity.

In some embodiments, the invention provides analogs of soluble CTLA4-Igmolecules, which have additional glycosylation sites. In otherembodiments, the invention provides analogs of solubleCTLA4^(A29YL104E)-Ig molecules, which have additional glycosylationsites. Additional glycosylation sites provide attachment points foradditional carbohydrate structures that can be sialylated. Increasedsialic content can be lead to increased PK values, and/or increasedglycoprotein stability. Higher sialic acid content is beneficial. Invitro post-purification methods that use enzymes to add more sialicacids can be performed to produce further embodiments of the CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules of the invention.

The embodiments of the invention include any one range disclosed hereinin combination with any one or more ranges disclosed herein. Theembodiments of the invention include any one characteristic or propertyof CTLA4-Ig disclosed herein in combination with any one or morecharacteristics or properties of CTLA4-Ig disclosed herein.

Methods for Analyzing and Isolating CTLA4-Ig and CTLA4^(A29YL104E)-IgGlycoproteins

The following methods described herein can be used to distinguish,identify, or isolate particular CTLA4-Ig or CTLA4^(A29YL104E)-Igmolecule populations on the basis of various sugar profiles, includingbut not limited to a population's average molar ratio of moles aminomonosaccharides and/or neutral monosaccharides and/or sialic acids permole CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules or dimer.

A glycoprotein that is secreted from cultured cells can be isolated fromthe culture medium or supernatant. The glycoprotein produced by thecells is collected, recovered, isolated, and/or purified, orsubstantially purified, as desired, at the end of the total cell cultureperiod using isolation and purification methods as known and practicedin the art or as described herein. In one embodiment, a glycoprotein ofthe invention, which is expressed by the cell but not secreted by thecell, can still be recovered from the cells, e.g., via making celllysates and isolating the glycoprotein, and/or using methods that areknown and practiced in the art, and as further described below.

The glycoprotein produced by the cell culture processes of thisinvention comprises complex carbohydrates that can be analyzed byvarious techniques of carbohydrate analysis. For example, techniquessuch as lectin blotting, well-known in the art, reveal proportions ofterminal mannose, or other sugars such as galactose. Termination ofmono-, bi-, tri-, or tetra-antennary oligosaccharide by sialic acids canbe confirmed by release of sugars from the protein using anhydroushydrazine or enzymatic methods and fractionation of oligosaccharides byion-exchange chromatography, size exclusion chromatography, or othermethods that are known in the art.

There are two main types of glycosidic linkages found in glycoprotiens,N-and O-linked. N-glycosylations are created by a covalent link of theglycan to the amide nitrogen of an asparagine residue. O-glycosidiclinkages are created by the covalent linkage of the hydroxyl group ofserine, threonine, hydroxylysine or hydroxyproline to the glycan. Thecarbohydrate moieties of glycoproteins are involved in numerousmolecular recognition phenomena, including host-pathogen interactions,clearance from serum and targeting of different tissues. With respect toCTLA4-Ig and CTLA4^(A29YL104E)-Ig molecules, carbohydrate moieties canat least affect binding between CTLA4-Ig molecules and CD80 or CD86, orbetween CTLA4^(A29YL104E)-Ig molecules and CD80 or CD86.

Carbohydrate structures typically occur on the expressed protein asN-linked or O-linked carbohydrates. The N-linked and O-linkedcarbohydrates differ primarily in their core structures. N-linkedglycosylation refers to the attachment of the carbohydrate moiety viaGlcNAc to an asparagine residue in the peptide chain. In one embodiment,the N-linked carbohydrates all contain a commonMan1-6(Man1-3)Man_(β)1-4Glc-Nac_(β)1-4GlcNac_(β)-R core structure, whereR in this core structure represents an asparagine residue. The peptidesequence of the protein produced will contain an asparagine-X-serine,asparagine-X-threonine, and asparagine-X-cysteine, wherein X is anyamino acid except proline.

In contrast, O-linked carbohydrates are characterized by a common corestructure, which contains GalNAc attached to the hydroxyl group of athreonine or serine. Of the N-linked and O-linked carbohydrates, themost important are the complex N-and O-linked carbohydrates. Suchcomplex carbohydrates contain several antennary structures. The mono-,bi-, tri, -, and tetra-, antennary structures are important for theaddition of terminal sialic acids. Such outer chain structures providefor additional sites for the specific sugars and linkages that comprisethe carbohydrates of the protein products.

Therapeutic glycoproteins are often produced using recombinant DNA cellculture techniques. Protein glycosylation distributions in cell culturecan be affected by variations in pH, cell density, nutrientconcentrations, and metabolite concentrations. The sensitivity of glycandistributions to environmental effects makes it necessary to carefullymonitor the glycan distribution during product development andproduction in order to ensure that a reproducible product ismanufactured.

The development of recombinant-derived glycoproteins for therapeutic usehas led to an increasing demand for methods to characterize and profiletheir carbohydrate structures. Oligosaccharide mapping has been usedduring initial characterization of recombinant proteins for comparisonto the native protein, to identify oligosaccharide structures present,to monitor consistency of oligosaccharide composition, to evaluatechanges that can result from alteration in cell culture or productionprocess, and to identify changes in glycosylation that occur as a resultof expression in different cell lines.

A variety of techniques are available to evaluate carbohydratestructural distributions. These include gel-filtration, chromatographicand electrophoretic separation techniques coupled with a wide range ofdetection techniques. If sample amounts are limited, the glycoproteinsare often derivatized with fluorescence reagents such as 2-aminobenzoicacid and 2-aminopyridine in order to improve detection. However,derivatization and purification of the derivatives can be timeconsuming. When sample size is not an issue, direct evaluation ofcarbohydrate structural distributions is possible.

Analysis of Oligosaccharide Content of a Glycoprotein

A particular glycoprotein can display heterogeneity of carbohydrates.Heterogeneity can be seen at several levels: glycosylation sites canvary from completely occupied to unoccupied, and any specific site canbe populated with many different oligosaccharide structures, whereineach structure can be modified by sialic acid molecules, such as NANA orNGNA.

The carbohydrate content of the protein of the present invention can beanalyzed by methods known in the art, including methods described in theExamples herein. Several methods are known in the art for glycosylationanalysis and are useful in the context of the present invention. Thesemethods provide information regarding the identity and the compositionof the oligosaccharide attached to the produced peptide. Methods forcarbohydrate analysis useful in connection with the present inventioninclude, but are not limited to, lectin chromatography; high performanceanion-exchange chromatography combined with pulsed amperometricdetection (HPAEC-PAD), which uses high pH anion exchange chromatographyto separate oligosaccharides based on charge; NMR; Mass spectrometry;HPLC; porous graphitized carbon (GPC) chromatography.

Methods for releasing oligosaccharides are known. These methodsinclude 1) enzymatic methods, which are commonly performed usingpeptide-N-glycosidase F/endo-α-galactosidase; 2) β-elimination methods,using a harsh alkaline environment to release mainly O-linkedstructures; and 3) chemical methods using anhydrous hydrazine to releaseboth N-and 0-linked oligosaccharides. Methods for analysis can comprisethe following steps: 1. Dialysis of the sample against deionized waterto remove all buffer salts, followed by lyophilization. 2. Release ofintact oligosaccharide chains with anhydrous hydrazine. 3. Treatment ofthe intact oligosaccharide chains with anhydrous methanolic HCl toliberate individual monosaccharides as O-methyl derivatives. 4.N-acetylation of any primary amino groups. 5. Derivatization to yieldper-O-trimethylsilyl methyl glycosides. 6. Separation of thesederivatives by capillary gas-liquid chromatography (GLC) on a CP-SIL8column. 7. Identification of individual glycoside derivatives byretention time from the GLC and mass spectroscopy, compared to knownstandards. 8. Quantification of individual derivatives by FID with aninternal standard (13-O-methyl-D-glucose).

The presence of neutral and amino sugars can be determined by using highperformance anion-exchange chromatography combined with pulsedamperometric detection (HPAEC-PAD Carbohydrate System; Dionex Corp.).For instance, sugars can be released by hydrolysis in 20% (v/v)trifluoroacetic acid at 100° C. for 6 hours. Hydrolysates are then driedby lyophilization or with a Speed-Vac (Savant Instruments). Residues arethen dissolved in 1% sodium acetate trihydrate solution and analyzed onan HPLC-AS6 column (as described by Anumula et al., 1991, Anal.Biochem., 195:269-280).

Alternatively, immunoblot carbohydrate analysis can be performed. Inthis procedure protein-bound carbohydrates are detected using acommercial glycan detection system (Boehringer), which is based on theoxidative immunoblot procedure described by Haselbeck et al. (1993,Glycoconjugate J., 7:63). The staining protocol recommended by themanufacturer is followed except that the protein is transferred to apolyvinylidene difluoride membrane instead of a nitrocellulose membraneand the blocking buffers contain 5% bovine serum albumin in 10 mM Trisbuffer, pH 7.4, with 0.9% sodium chloride. Detection is carried out withanti-digoxigenin antibodies linked with an alkaline phosphate conjugate(Boehringer), 1:1000 dilution in Tris buffered saline using thephosphatase substrates, 4-nitroblue tetrazolium chloride, 0.03% (w/v)and 5-bromo-4 chloro-3-indoyl-phosphate 0.03% (w/v) in 100 mM Trisbuffer, pH 9.5, containing 100 mM sodium chloride and 50 mM magnesiumchloride. The protein bands containing carbohydrate are usuallyvisualized in about 10 to 15 minutes.

Carbohydrates associated with protein can also be cleaved by digestionwith peptide-N-glycosidase F. According to this procedure the residue issuspended in 14 μL of a buffer containing 0.18% SDS, 18 mMbeta-mercaptoethanol, 90 mM phosphate, 3.6 mM EDTA, at pH 8.6, andheated at 100° C. for 3 minutes. After cooling to room temperature, thereaction mixture is divided into two approximately equal parts. Onepart, which is not treated further, serves as a control. The other partis adjusted to about 1% NP-40 detergent followed by the addition of 0.2units of peptide-N-glycosidase F (Boehringer). Both parts are warmed at37° C. for 2 hours and then analyzed by SDS-polyacrylamide gelelectrophoresis.

Glycan mapping of glycoproteins is becoming increasingly accepted. Themethodology described herein allows for rapid characterization ofoligosaccharides in terms of glycan type, extent of sialylation andnumber of branches on the non-reducing end of the carbohydrates. Thus,in certain embodiments, the invention provides CTLA4-Ig populationscharacterized by particular oligosaccharide profiles. Oligosaccharideprofiling is typically done by chromatographic separation ofoligosaccharides followed by detection and relative quantitation. Analternative to chromatographic profiling is the direct analysis ofoligosaccharides by ESI infusion after online desalting.

Oligosaccharide profiling by PGC can be been used to characterize theN-linked oligosaccharides from CTLA4-Ig molecules. There are 31structural classes of oligosaccharides identified from CTLA4-Igmolecules (SEQ ID NO:2), including a structural class containingO-acetylated sialic acid groups. Structural class verification isachieved through the use of MS/MS and positive ion mode MS. Relativequantitation of structural classes is possible through integration ofthe UV trace at 206 nm. Comparison of the subpopulation profiles fromindividual N-link sites is known, revealing significant populationdifferences between N-link sites. Oligosaccharide profiling using PGCprovides a convenient information rich alternative to the moretraditional profiling methods such as HPAEC.

N-linked Structures in CTLA4-Ig Molecules Comprising Monomers of SEQ IDNO:2

There are three N-linked glycosylation sites per chain (i.e., permonomer) on a CTLA4-Ig multimer or dimer, wherein the monomer has asequence from SEQ ID NO:2, for example: (i) 26-383 of SEQ ID NO:2, (ii)26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQID NO:2, (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2). Thevariations in glycosylation by site are analyzed by isolating peptidefragments containing N-linked glycans from a tryptic digest of theprotein. The N-linked glycosylation sites on the protein are located atAsn^(102,) Asn¹³⁴ and Asn²³³, contained in tryptic fragments 5, 7, and14, respectively. Enzymatic release of N-linked oligosaccharides fromthe isolated peptide fragments, followed by PGC profiling of thereleased oligosaccharides results in the profiles shown in FIG. 12. Itis clear from the profile of the glycans released from Asn²³³ (Trypticfragment 14, T14) that the oligosaccharide population is enriched in theasialo structures (structures have no sialic acids). Oligosaccharideprofiles from the glycans attached at Asn¹⁰² and Asn¹³⁴ (T5 and T7)contain the bulk of the sialylated structures

Isolated oligosaccharides released from glycoprotein are directlyinjected into the porous graphitized carbon LC/UV/MS system. FIGS. 13and 14 show the TIC and UV chromatograms of a typical PGC profilesgenerated by acetonitrile gradients containing acidic and basicadditives. In most cases, the mass spectra from a single chromatographicpeak contain mass peaks for a single oligosaccharide. Thirtyoligosaccharide structural classes are identified from the TFAcontaining elution profile. Only sixteen oligosaccharide structuralclasses are identified from the NH₄OH containing elution profile. Withineach structural class there are variant structures containingsubstitution of N-glycolylneuraminic acid (NGNA) in place ofN-acetylneuraminic acid (NANA) as well as differing degrees of sialicacid acetylation. Although only qualitatative information can be gainedfrom comparison of the ion counts for the oligosaccharide classes, it isapparent that the major structural classes within each of the fourdomains are P2100, P2111, P2122, and P3133. This is consistent with theintegration values obtained from the UV trace at 206 nm. Furtherstructural verification can be obtained from the positive ion massspectrogram. Positive ion mode ionization promotes in sourcefragmentation of oligosaccharides, mainly at the glycosidic bonds.Because there is good separation of oligosaccharides, as determined bythe negative ion mass spectra, the fragmented spectra from the positiveion mode mimic the positive ion MS/MS spectra. Domain III (di-sialylatedstructures) contains a significant amount of the O-acetylated structureP2122-Ac. Positive ion m/s data supports O-acetylation of one of thesialic acids on the structure. The most common O-acetylation site ofsialic acid residues are at the C-7 and C-9 positions (Shi WX, ChammasR., Varki A., J. Biol. Chem. 271 (1996) 15130-15188). At physiologicextracellular pH, O-acetyl esters at C-7 spontaneously migrate to C-9.The most likely O-acetlylation site is therefore C-9.

Analysis of N-linked Oligosaccharide Content: The analytical techniquescan comprise cleavage and isolation of N-linked oligosaccharides bycolumn chromatography, which in a non-limiting embodiment uses aHypercarb column. Glycans subjected to Hypercarb chromatography areisolated and can be analyzed by HPAEC-PAD which analysis determines thetypes of carbohydrates that modify a particular glycoprotein. Analyticalcharacterization of the N-linked oligosaccharides can also be achievedby Liquid Chromatography/Mass Spectrometry (LC/MS) using a PorousGraphitized Carbon (PGC). Carbohydrate analysis can also includetrypsin, Asp-N, and Trypsin/Chymotrypsin peptide mapping to determinethe peptides, which comprise carbohydrate structures.

N-linked oligosaccharide structures can be analyzed using a series oforthogonal mass spectrometry and HPAEC-PAD techniques (see Examples).These techniques include several endopeptidase cleavages followed byLC/MS/MS analysis. With respect to CTLA4-Ig monomers having a sequencefrom SEQ ID NO:2, the three major sites of N-linked glycosylation werecharacterized using LC/MS and LC/MS/MS electrospray ionization and themajor structures at each N-link site were determined. These data aresummarized in FIG. 9. There are at least three major attachment pointsfor N-linked oligosaccharides at Asn¹⁰², Asn¹³⁴, and Asn²³³. Inaddition, Asn²³³ is found to contain a population of N-linked structuresthat contained no sialic acid groups occurring about 80% of the time.

N-linked oligosaccharide structures of CTLA4-Ig determined by LC/MS ofthe glycopeptides, LC/MS of the oligosaccharides, and HPAEC-PAD: TheN-linked carbohydrates are associated with a consensus sequence motif ofAsn-X-Ser/Thr. This sequence appears three times on CTLA4-Ig monomerchains having one of the following sequences: (i) 26-383 of SEQ ID NO:2,(ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 ofSEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2.The consensus sequence motif appears in SEQ ID NO:2 at: Asn¹⁰² Leu¹⁰³Thr¹⁰⁴; Asn¹³⁴ Gly¹³⁵ Thr¹³⁶; and Asn²³³ Ser²³⁴ Thr²³⁵. Based on theconsensus sequence, there are six N-linked carbohydrate sites per dimermolecule that is formed of any one or two of the following monomersequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii)27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 of SEQ IDNO:2, and (vi) 25-383 of SEQ ID NO:2.

N-linked carbohydrates can be of three general varieties: high-mannose,hybrid and/or complex. A LC/MS technique for the glycopeptide analysiswas developed. Trypsin endoproteolytic cleavage of monomers (having oneof the sequences (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2,(iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v) 25-382 ofSEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2) result in three peptidesthat contain N-linked glycosylation. All three N-linked sites arepopulated with carbohydrate structures. Tryptic fragment T5corresponding to amino acids 65-109 of SEQ ID NO:2 contains aglycosylation on Asn¹⁰². Tryptic fragment T7 corresponding to aminoacids 120-154 of SEQ ID NO:2 contains a glycosylation on Asn¹³⁴. Trypticfragment T14 corresponding to amino acids 229-237 of SEQ ID NO:2contains a glycosylation on Asn²³³.

In order to determine the specific types of glycosylation on each site,carbohydrates were obtained from each specific site by increasing thescale of protein digestion and separation followed by collection of theT5, T7, and T14 peptides. The tryptic peptide peaks of interest weretreated with PNGase F and processed for analysis by LC/MS on a Hypercarbcolumn. Results showed that a heterogeneous population of complex bi-,tri-, and tetra-antennary structures at each site. These can be seen inFIG. 12 where the chromatography separates the sugars into five domains:asialo, mono-sialo, di-sialo, tri-sialo, and tetra-sialo structures(referred to as Domains I, II, III, IV, and V, respectively). Achromatogram (FIG. 12 panel A) for the Asn¹⁰² (T5) carbohydratesillustrates a series of mono-and di-sialo structures at the site. Achromatogram (FIG. 12 panel B) for Asn¹³⁴ (T7) illustrates two maindi-sialo structures with a population of mono-sialo structures. Achromatogram (FIG. 12 panel C) for Asn²³³ (T14) illustrates littlesialylation. For each of the N-linked carbohydrate sites, a MS spectrumand corresponding structure is shown for the major peak in eachchromatogram (see FIG. 12, panels E, F, H). In FIG. 12, panel D, thetotal N-linked carbohydrate profile of CTLA4-Ig is shown in thechromatogram. The mass and structures of selected peaks are listed inTable 1. The oligosaccharide LC/MS data were supported by in-depthanalysis of the peptide map. Asn¹⁰² (T5 peptide) has the greatest degreeof carbohydrate heterogeneity ranging from bi-antennary, non-sialylatedstructures to tetra-antennary, tetra-sialylated structures. Asn¹³⁴ (T7peptide) contains primarily bi-antennary structures. This site containsmuch less heterogeneity than the Asn¹⁰² site. The Asn²³³ (T14 peptide)site contains little sialylation. A third analytical technique,HPAEC-PAD, was also employed to support the two orthogonal LC/MSfindings.

TABLE 1 The major N-linked structures and selected minor complexstructures observed using LC/MS methods Theo- Actual reticalDeconvoluted Structure Mass Mass (GlcNAc)₄ (Fuc)1 (Man)₃ 1462  1575*(GlcNAc)₄ (Fuc)1 (Man)₃ (Gal)₁ 1624  1737* (GlcNAc)₄ (Fuc)1 (Man)₃(Gal)₂ 1786  1899* (GlcNAc)₄ (Fuc)1 (Man)₃ (Gal)₁ (NeuAc)₁ 1916 1916(GlcNAc)₄ (Fuc)1 (Man)₃ (Gal)₂ (NeuAc)₁ 2077 2077 (GlcNAc)5 (Fuc)1(Man)₃ (Gal)₃ (NeuAc)₁ 2443 2442 (GlcNAc)₄ (Fuc)1 (Man)₃ (Gal)₂ (NeuAc)₂2369 2368 (GlcNAc)₅ (Fuc)1 (Man)₃ (Gal)₃ (NeuAc)₂ 2734 2734 (GlcNAc)₅(Fuc)1 (Man)₃ (Gal)₃ (NeuAc)₃ 3025 3025 (GlcNAc)₆ (Fuc)1 (Man)₃ (Gal)₃(NeuAc)₃ 3388 3388 (GlcNAc)₆ (Fuc)1 (Man)₃ (Gal)₃ (NeuAc)₄ 3680 3680*The asialo species are detected as TFA adducts.

The population of total N-linked carbohydrates was analyzed usingHPAEC-PAD. The data obtained by this method are listed in Tables 2 and3. In Table 2, the relative area percentages of asialo to tri-sialodomains are listed within each site (Asn¹⁰², Asn¹³⁴, and Asn²³³ of SEQID NO:2). In Table 3, the oligosaccharide domain area percentages arelisted as a fraction of the entire population of oligosaccharides.

TABLE 2 The area percentages for each domain observed by the HPAEC-PAD Nlinked Asialo Mono Di Tri N¹⁰² 27 37 25 11 N¹³⁴ 25 38 28 8 N²³³ 82 12 51

TABLE 3 The area percentages for each domain expressed as weightedaverage on Table 2 data set. N linked Asialo Mono Di Tri N¹⁰² 9 12 8 4N¹³⁴ 8 13 9 3 N²³³ 28 4 2 0 Total/Molecule 45 29 19 7Assuming full glycosylation.

N-linked oligosaccharide structures of CTLA4^(A29YL104E)-Ig moleculesdetermined by LC/MS of the glycopeptides, LC/MS of the oligosaccharides,and HPAEC-PAD: The N-linked carbohydrates are associated with aconsensus sequence motif of Asn-X-Ser/Thr. This sequence appears threetimes on CTLA4^(A29YL104E)-Ig monomer chains having one of the followingsequences: (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii)27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ IDNO:4, and (vi) 25-383 of SEQ ID NO:4. The consensus sequence motifappears in SEQ ID NO:4 at: Asn¹⁰² Leu¹⁰³ Thr¹⁰⁴; Asn¹³⁴ Gly¹³⁵ Thr¹³⁶;and Asn²³³ Ser²³⁴ Thr²³⁵. Based on the consensus sequence, there are sixN-linked carbohydrate sites per dimer molecule that is formed of any oneor two of the following monomer sequences: (i) 26-383 of SEQ ID NO:4,(ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv) 27-382 ofSEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4.

N-linked carbohydrates can be of three general varieties: high-mannose,hybrid and/or complex. A LC/MS technique for the glycopeptide analysiswas developed. Trypsin endoproteolytic cleavage of monomers (having oneof the sequences (i) 26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4,(iii) 27-383 of SEQ ID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 ofSEQ ID NO:4, and (vi) 25-383 of SEQ ID NO:4) result in three peptidesthat contain N-linked glycosylation (See Table 25 in EXAMPLE 22). Allthree N-linked sites are populated with carbohydrate structures. Trypticfragment T5 corresponding to amino acids 65-109 of SEQ ID NO:4 containsa glycosylation on Asn¹⁰². Tryptic fragment T7 corresponding to aminoacids 120-154 of SEQ ID NO:4 contains a glycosylation on Asn¹³⁴. Trypticfragment T14 corresponding to amino acids 229-237 of SEQ ID NO:4contains a glycosylation on Asn²³³ (See Table 25 in EXAMPLE 22).

In order to determine the specific types of glycosylation on each site,carbohydrates were obtained from each specific site by increasing thescale of protein digestion and separation followed by collection of theT5, T7, and T14 peptides. The tryptic peptide peaks of interest weretreated with PNGase F and processed for analysis by LC/MS on a Hypercarbcolumn. Results showed that a heterogeneous population of complex bi-,tri-, and tetra-antennary structures at each site. These can be seen inFIG. 15 where the chromatography separates the sugars into four domains:asialo, mono-sialo, di-sialo, and tri-sialo structures (referred to asDomains I, II, III, and IV respectively). The characteristics of acarbohydrate profile that can be analyzed and compared betweenglycosylated molecules, or populations or compositions comprisingglycosylated molecules include peak area percent, domain area percent,valley-to-valley distance, or peak-to-peak distance.

LC/MS Characterization of CTLA4-Ig N-Linked Oligosaccharides

LC/MS porous graphitic carbon (PGC) chromatography is a method forprofiling N-linked oligosaccharides can provide several advantages overthe High pH Anion Exchange Chromatography (HPAEC). Some of theseadvantages include: Direct profiling from digest mixtures whichminimizes sample loss and degradation; direct MS interface provides amethod for rapid characterization and analysis of oligosaccharides;increased resolution through PGC chromatography permits both theinter-domain comparisons as well as more subtle intra-domain analysis.

The LC/MS PGC method allows for rapid profiling and characterization ofoligosaccharides in terms of glycan type, and determining the extent ofsialylation and branching on the non-reducing end of the carbohydrates.Then negative ion mode MS spectra produce data that is simple tointerpret, with minimal oligosaccharide fragmentation, while positivemode ionization allows for structural class verification. The methoddescribed here can be applied to whole digest mixtures of glycoproteins,as well as to previously isolated oligosaccharide samples without theneed for derivatization. The chromatographic mobile phases used allowfor collection of peaks from the profiles and concentration to dryness,without further manipulation for more detailed characterization. In oneembodiment the method is used to characterize CTLA4-Ig N-linkedoligosaccharides. Using the LC/MS PGC method, thirty-one distinctclasses of oligosaccharides can be identified on CTLA4-Ig moleculescomprised of monomers having sequences from SEQ ID NO:2, e.g., SEQ IDNO: 5, 6, 7, 8, 9, or 10.

High pH anion exchange chromatography (HPAEC) has been used extensivelyto profile oligosaccharides released from glycoproteins without the needfor derivitization. The high resolution of HPAEC and the fact that theseparation is influenced by the type of sugar residue present, type oflinkage and the size of the glycan are reasons for the widespread use ofthe technique. The dominating factor in separation is charge, highlycharged oligosaccharides eluting later than less charged glycans.Chromatographic profiles are often divided into domains defined by thenumber of charged species, typically sialic acid residues, on theglycans (FIG. 16).

To obtain more information about the structure of the unknownoligosaccharides the HPAEC peaks can be collected, desalted andcharacterized by MS and/or NMR. One consideration to HPAEC-PAD profilingof oligosaccharide distributions is the variability inherent to thedetection mode. Electrochemical cell aging and electrode surface foulingresult in profile variability. It has also been reported thatoligosaccharide structures and the degree of sialylation can causevariability among detection cells when using HPAEC with pulsedamperometric detection (HPAEC-PAD). This variability can affectquantitative and relatively quantitative results used to evaluate theeffect of process changes or determine batch to batch consistency.Because of its speed and specificity, mass spectrometry (MS) has gainedpopularity as a technique for assessment of oligosaccharide profiling ofglycoprotiens. Although MS profiles cannot be used to directly determineanomeric configuration or branching patterns, MS data can be used toidentify structural classes and detect qualitative changes in glycoformdistributions.

The porous graphitized carbon (PGC) chromatographic profiling method forenzymatically released N-linked oligosaccharides uses both ultraviolet(UV) and mass spectroscopic (MS) detection to profile and characterizeN-linked oligosaccharides, either directly from enzymatic digestmixtures or from isolated oligosaccharides. This method can be used toprofile and characterize oligosaccharide released from CTLA4-Igglycoproteins. The LC/MS PGC method can evaluate the consistency of theoligosaccharide distributions resulting from the production process, aswell as any changes in the oligosaccharide distributions resulting fromprocess modifications. In a chromatographic microanalysis of N-linkedoligosaccharides of CTLA4-Ig molecules, enzymatically released N-linkedoligosaccharides can be readily separated by the PGC column in order ofincreasing sialylation and increasing size. The range of structurespresent and the relative amounts of each class of structure aredetermined through a combination of MS and UV analysis (Example 3).

To optimize the LC/MS PGC method, optimization of mass spectralconditions can be useful. Optimization can include a set of surfacemapping experiments in order to evaluate the effects of solventcomposition and MS ionization parameters on oligosaccharide detection.Solvent composition parameters for evaluation comprise percentageacetonitrile (by volume) and eluent additives (trifluoroacetic acid andammonium hydroxide). MS ionization parameters evaluated for evaluationinclude the desolvation temperature, capillary voltage and cone voltagesettings for the electrospray source.

The ionization parameters can play a significant role in signalresponse. The model resulting from the surface mapping determination wasused to set ionization parameters during the chromatographicdetermination. Higher values for both desolvation temperature and conevoltage result in greater response. The capillary voltage optimum variesdepending on the eluent additive, the TFA containing solvent systemhaving a slightly higher optimal capillary voltage. The factor with thelargest effect is the volume percentage of acetonitrile, higheracetonitrile content resulting in higher responses.

Porous Graphitized Carbon Chromatography

Porous graphitized carbon (PGC) has been used for solid phase extractiondesalting of oligosaccharides. PGC has also been known as an effectivechromatographic media for oligosaccharide separation under both acidicand basic elution conditions. Chromatography conditions for both acidicand basic profiling of enzymatically released oligosaccharides fromCTLA4-Ig molecules having monomer sequences from SEQ ID NO:2 aredeveloped. Each condition is compatible with both UV and MS detection.As was observed in the infusion experiments, the acidic elutionconditions result in higher MS sensitivity than the basic conditions.The MS response for neutral oligosaccharides eluted under acidicconditions, detected as TFA adducts, are five to nine times theintensity of the corresponding peak eluted under basic conditions. Thedifference in signal response is less dramatic for the acidicoligosaccharides, averaging three times the signal response formonosialylated glycans and equal signal response for di-sialylatedglycans. The increased number of peaks in the TFA eluted chromatogram(FIGS. 13A-13B) compared to the NH₄OH eluted chromatogram (FIG. 14A-14B)is a result of separation of anomeric forms of oligosaccharides.Collection and concentration of individual peaks eluted from the TFAgradient result in splitting of the single peak into two peaks ofidentical mass upon re-injection. Basic elution of the oligosaccharidesfrom the PGC column results in a simpler profile (FIG. 14A-14B). Thebasic elution conditions do not result in complete anomeric separation,however significant peak broadening is observed. The peaks resolutioncan be increased by increasing the column temperature, which willaccelerate the interchange of anomeric forms (Itoh S., et al., J.Chromatogr. A. 2002 968(1-2), 89-100). However, the sensitivity (ioncount) for the detected oligosaccharides remains reduced compared to theacidic elution conditions. It has been reported that addition of saltssuch as ammonium acetate can increase sensitivity. (Churms SC, J.Chromatogr. A. 500 (1990) 555-583.)

Addition of ammonium acetate, ammonium trifluoroacetate or ammoniumformate results in increased response but also results in asymmetricpeak broadening. The resulting peak broadening and potentialinterference of the added salt with UV detection made salt addition anunattractive option. An alternative means of eliminating anomericseparation is to reduce the oligosaccharides to the correspondingalditols.

Higher sensitivity and chromatographic resolution make the acidicelution conditions useful for oligosaccharide profiling. A particularprofiling system consists of a Luna C18 column coupled through twodual-position six port valves to the Hypercarb 5 μM column (100×4.6 mm).The Hypercarb column is coupled to a UV detector (Waters 2996 PDA) inseries with a Q-ToF Micro (Micromass) with a standard ESI probe. Throughappropriate switch control, prepurified CTLA4-Ig samples can be profiledusing the Hypercarb column alone, or digest mixtures can be profiled bydirect injection of the digest mixture onto the Luna C18 in series withthe Hypercarb column. Typically, profiles are obtained from the N-linkedoligosaccharides released from 10 to 20 nmoles of protein.

In certain embodiments, the invention provides a population of CTLA4-Igmolecules that have a chromatogram according to any one or more of thechromatograms having representative peaks. Representativeoligosaccharide profile chromatograms for CTLA4-Ig molecules havingmonomers with sequences from SEQ ID NO:2 are shown in FIG. 12, FIGS.13A-13B, FIGS. 14A-14B (PGC), FIG. 16 (HPAEC/PAD), and FIGS. 17A-17B.Both of these chromatographic profiles can be broken down into fourdistinct domains containing oligosaccharide structures with increasingdegrees of sialylation in the later eluting domains. The PGCchromatographic system allows for direct interface with a mass detector.The mass resolution and signal to noise ratios are acceptable even foroligosaccharides which are present in low percentages. Chromatographicresolution of individual oligosaccharide structures appears greater inthe PGC chromatographic separation as compared to the HPAEC.

Collecting peaks from the HPAEC method requires desalting and the highpH employed introduces the possibility of peeling reactions that couldinterfere with accurate structural identification of peaks. Because thechromatographic conditions used with PGC chromatography are free ofsalts, the eluted oligosaccharide peaks can be collected andconcentrated with minimal manipulation. This allows for the collectionand concentration of eluted peaks, followed by injection of thecollected oligosaccharides onto the HPAEC system. Re-injection of thecollected oligosaccharides onto a HPAEC-PAD system allows for structuralassignment of some of the peaks present in the HPAEC profile (FIG. 16).Due to incomplete peak resolution on the anion exchange column, not allof the isolated peaks could be mapped to the HPAEC profile.

The profiles resulting from direct injection of digest mixtures andthose for isolated oligosaccharides from the same protein sample are notidentical. The profiles resulting from direct injection (FIGS. 17A-17B)have different anomer ratios suggesting that the concentration ofcollected oligosaccharides is resulting in increased anomerization. Moreimportantly, the profile resulting from direct injection contains apeak, which corresponds to a tetra-sialylated structure. This structureis not identified in the profile of the collected and isolatedoligosaccharides. In addition to shortened assay time, profilingdirectly from digest mixtures can result in a more accuraterepresentation of the oligosaccharide distribution, by avoiding glycandegradation during collection and concentration.

Relative Quantitation

The surface mapping performed on infusion samples indicates that thevolume percentage of acetonitrile has a significant effect on theionization efficiency of eluting oligosaccharides. The dependence ofsignal intensity on acetonitrile content in the mobile phase makesrelative quantitation of oligosaccharides by MS dependent on theretention time of the eluting peak. Variations in column condition caneffect elution times on PGC columns. For this reason, it would bedifficult to obtain consistent relative quantitation from the ionchromatogram elution profile. The UV trace at 206 nm should not beaffected by the solvent composition to the same extent as the ion trace.The relative quantitation was performed using the UV trace, the iontrace was used for characterization and qualitative comparisons only.Replicate injections for oligosaccharides isolated from a singleglycoprotein lot resulted in percent relative standard deviations (%RSD) of less than 4% for each of the four oligosaccharide domainsquantified.

O-linked Structures in CTLA4-Ig Molecules Comprising Monomers of SEQ IDNO:2

In addition to the N-linked carbohydrates, CTLA4-Ig molecules cancontain O-linked carbohydrates. The O-linked oligosaccharide structurescan be analyzed using a series of orthogonal mass spectrometrytechniques. These techniques include several endopeptidase cleavagesfollowed by LC/MS/MS analysis.

With respect to CTLA4-Ig molecules formed of monomers having a sequencefrom SEQ ID NO: 5, 6, 7, 8, 9, or 10, the two major sites of O-linkedglycosylation were characterized using exact mass electrosprayionization and the major structures at each 0-link site were determined.These data are summarized in FIG. 9. Data are consistent with therebeing three major O-linked structures: (GalNAc)₁ (Gal)₁ (NeuAc)₁;(GalNAc)₁ (Gal)₁ (NeuAc)₂; (GalNAc)₁ (GlcNac)₁ (Gal)₂ (NeuAc)₂. Eachstructure is observed in differing amounts on each site. These amountsare relatively quantitative and represent data obtained from multipleanalyses. The O-linked oligosaccharides contribute a substantial amountof sialic acid to CTLA4-Ig. There are two major O-linked oligosaccharideattachment points per chain. The primary site of occurrence for O-linkedoligosaccharides is Ser¹⁶⁵, which is occupied in about 95% of the time.The secondary site of occurrence for O-linked oligosaccharides isSer¹⁵⁵¹¹⁵⁶ which is occupied=25% of the time. The orthogonal datapresented herein provides an overview of the predominant carbohydratestructures present on such CTLA4-Ig molecules and is summarized in FIG.9.

In general, the O-linked carbohydrates have far greater heterogeneity ofstructure than are present in N-linked carbohydrates. In addition, thereis no consensus sequence for 0-link attachment. Thus, a series oforthogonal techniques were developed for use in the structuralcharacterization of the O-linked oligosaccharides: LC/MS intact analysisand LC/MS glycopeptide analysis.

Based on Edman degradation and MALDI, an 0-link site was reported to beSer¹⁶⁵ (with respect to SEQ ID NO:2). To obtain direct data for thepresence of Ser¹⁶⁵ glycosylation, MS/MS sequencing using b′ and y″ ionseries on the T9 peptide (see Table 4 and Table 5) was performed. Table4 lists the ion series for the T9 peptide in four different states ofglycosylation. In all four states, the b′ ion series, b1 . . . b6 ionsare in agreement. However, the b′ ion series, b7 . . . b_(max) vary bythe different glycosylation states at b7 (Ser¹⁶⁵). As a confirmation,the corresponding y″ ion series is reported. In all four y″ ion series,the y1 . . . y19 ions are in complete agreement. However, the y″ ionseries, y20 . . . ymax vary by the different glycosylation states at y20(Ser¹³⁹). The b′ and y″ ion series taken together support theimplication of Edman sequencing that Ser¹³⁹ is the primary site ofO-linked glycosylation on the T9 peptide. T9 is a peptide that containsseveral serine and theronine residues.

Table 4 presents LC/MS/MS b′ and y″ ions for the T9 peptide with andwithout the O-linked ladder of (GalNAc)₁ (Gal)₁ (NeuAc)₁. The b′ ionseries are identical for all spectra until b7 where the spectrum thendiffers by the O-linked carbohydrate structure listed above each series.The y′ ion series are identical for all spectra until y19 where thespectrum then differs by the O-linked carbohydrate structure listedabove each series.

TABLE 4 LC/MS/MS b′ and y″ ions for the T9 peptide. T9-GalNAc-Gal-NeuAcT9-GalNac-Gal T9-GalNac T9 b′ y″ b′ y″ b′ y″ b′ y″ 1 Thr 102.1 — 1 Thr102.1 — 1 Thr 102.1 — 1 Thr 102.1 — 26 26 26 26 2 His 239.1 3243.6 2 His239.1 2952.5 2 His 239.1 2790.5 2 His 239.1 2587.4 25 25 25 25 3 Thr340.2 3106.6 3 Thr 340.2 28152.5  3 Thr 340.2 2653.4 3 Thr 340.2 2450.324 24 24 24 4 Ser 427.2 3005.5 4 Ser 427.2 2714.4 4 Ser 427.2 2552.4 4Ser 427.2 2349.3 23 23 23 23 5 Pro 524.2 2918.5 5 Pro 524.2 2627.4 5 Pro524.2 2465.3 5 Pro 524.2 2262.3 22 22 22 22 6 Pro 621.3 2821.4 6 Pro621.3 2530.3 6 Pro 621.3 2368.3 6 Pro 621.3 2165.2 21 21 21 21 7 Olk1364.6 2724.4 7 Oln 1073.52 2433.3 7 Oli 911.4 2271.2 7 Ser 708.3 2068.120 20 20 20 8 Pro 1461.6 1981.1 8 Pro 1170.5 1981.1 8 Pro 1008.5 1981.18 Pro 605.4 1981.1 19 19 19 19 9 Ala 1532.6 1884.1 9 Ala 1241.6 1884.1 9Ala 1079.5 1884.1 9 Ala 876.4 1884.1 18 18 18 18 10 Pro 1629.7 1813.0 101338.6 1813.0 10 1176.6 1813.0 10 973.5 1813.0 17 Pro Pro Pro 17 17 1711 1758.7 1716.0 11 1467.6 1716.0 11 1305.6 1716.0 11 1102.5 1716.0 GluGlu Glu Glu 16 16 16 16 12 1871.8 1586.9 12 1580.7 1586.9 12 1418.71586.9 12 1215.6 1586.9 Leu Leu Leu Leu 15 15 15 15 13 1984.9 1473.8 131693.8 1473.8 13 1531.8 1473.8 13 1328.7 1473.8 Leu Leu Leu Leu 14 14 1414 14 2041.9 1360.8 14 1750.8 1360.8 14 1588.8 1360.8 14 1385.7 1360.8Gly Gly Gly Gly 13 13 13 13 15 2099.0 1303.7 15 18070.9 1303.7 15 1645.81303.7 15 1442.7 1303.7 Gly Gly Gly Gly 12 12 12 12 16 Ser 2186.0 1246.716 1894.9 1246.7 16 1732.8 1246.7 16 1529.8 1246.7 11 Ser Ser Ser 11 1111 17 Ser 2273.0 1159.7 17 1981.9 1159.7 17 1819.9 1159.7 17 1616.81159.7 10 Ser Ser Ser 10 10 10

TABLE 5 O-linked glycopeptide fragments with the corresponding sequencenumbers, amino acid sequences and theoretical masses Amino Acid EnzymeSequence Sequence Unmodified Enzyme Fragment Fragment (SEQ ID NO: 2)Mass Trypsin T9 159-184 THTSPPSPAPELLG 2688.44 GSSVFLFPPKPK AspN D8150-156 DQEPKSS 790.36 Tryp/ N/A 159-171 THTSPPSPAPELL 1345.7 chrmo

The O-linked carbohydrate structures at Ser¹⁶⁵ represent a heterogeneouspopulation of three major species. In FIG. 18, the T9 glycopeptide isobserved in the deconvoluted spectrum. There is a base peak at 2689.2amu which is in agreement with the theoretical mass for this peptide of2689.11 amu. The spectrum illustrates three major O-linked structures.The spectrum illustrates the base peptide with a sugar ladder consistentwith the O-linked structure (GalNAc)₁ (Gal)₁ (NeuAc)₁. The magnifiedbold portion of the spectrum has been enhanced 10-fold and identifiestwo additional O-linked structures consistent with (GaNAc)₁ (Gal)₁(NeuAc)₂ and (GaNAc)₁ (GlcNAc)₁ (Gal)₂ (NeuAc)₂.

Mass spectrometry was used to assess the relative abundance of eachO-linked species. In FIG. 18, the (GalNAc)₁ (Gal)₁ (NeuAc)₁ glycan isobserved in a 10:1 ratio with the (GalNAc)₁ (Gal)₁ (NeuAc)₂ glycan andin a 30:1 ratio with the (GalNAc)₁ (G1cNAc)₁ (Gal)₂ (NeuAc)₂ glycan. Inone embodiment therefore, the invention provides a population comprisingCTLA4-Ig molecules that have a 10:1 ratio of (GalNAc)₁ (Gal)₁ (NeuAc)₁glycan to (GalNAc)₁ (Gal)₁ (NeuAc)₂ glycan. In another embodiment, theinvention provides a population comprising CTLA4-Ig molecules that havea 30:1 ratio of (GalNAc)₁ (Gal)₁ (NeuAc)₁ glycan to (GalNAc)₁ (G1cNAc)₁(Gal)₂ (NeuAc)₂ glycan. The (GalNAc)₁ (Gal)₁ (NeuAc)₂ glycan is observedin a ratio of 20:1 with the (HexNAc)₂ (Gal)₂ (NeuAc)₂ glycan. In anotherembodiment, the invention provides a population comprising CTLA4-Igmolecules that have a 20:1 ratio of the (GalNAc)₁ (Gal)₁ (NeuAc)₂ glycanto (HexNAc)₂ (Gal)₂ (NeuAc)₂ glycan. In another embodiment, theinvention provides a population of CTLA4-Ig molecules that comprise allof the said ratios in this paragraph. In addition, a negative ionelectrospray spectrum confirms these three predominant structures; therelative abundance of each is shown in FIG. 9.

With respect to CTLA4-Ig molecules comprising monomers of SEQ ID NO:2,in addition to the Ser¹⁶⁵ site, a second 0-link site is observed atSer¹⁵⁵ or Ser¹⁵⁶. This site is referred to as Ser^(155/156). The D8peptide containing Ser^(155/156) was generated from an AspN digestionand corresponds to amino acids 150-156 of SEQ ID NO:2. The peptide isseparated and detected by LC/MS. The spectrum (not shown herein) for theD8 0-linked glycopeptide shows a base peak of 790.2 amu that is inagreement with the theoretical mass of 790.8 amu. The spectrumillustrates the peptide ion and a series of ions which are consistentwith the structure, (GalNAc)₁ (Gal)₁ (NeuAc)₁. The peptide ispredominantly non-glycosylated; the glycosylated (GalNAc)₁ (Gal)₁(NeuAc)₁ species constitutes approximately 22% peak area.

The O-linked oligosaccharide structures of the CTLA4-Ig single chainwere characterized using a series of orthogonal mass spectrometrytechniques. These techniques include endopeptidase cleavages with LC/MSanalysis of the two predominant sites of O-linked glycosylation with theuse of electrospray ionization to determine the predominant structuresat each 0-link site. These data are summarized in FIG. 19. There can befour predominant O-linked structures: (GalNAc)₁(Gal)₁(NeuAc)₁;(GalNAc)₁(Ga)₁ (NeuAc)₂; (HexNAc)₂(Gal)₂(NeuAc)₂; (HexNAc)₂(Gal)₂,(NeuAc)₃. These structures are detected in differing amounts on eachsite. Greater than 95% of the CTLA4-Ig single chain has at least(HexNAc)₂ (Gal)₂ (NeuAc)₂.

Another assay was developed to confirm the O-linked carbohydrates andlook for less prevalent structures. This technique utilized trypsin andchymotrypsin co-digestion to produce a peptide confirmed by MS/MS to beTHTSPPSPAPELL (amino acids 159-171 of SEQ ID NO:2). This peptide allowedfor the identification of one monosialylated, two di-sialylated and onetri-sialylated O-linked species. A definitive structure has not beenelucidated for the tri-sialylated species, however two possibilities areproposed: a peptide containing a core 2 structure with 3 sialic acids ortwo core 1 structures present on two different amino acid residues.

A complementary technique, intact analysis by MS, was used to confirmthe presence of heterogeneous 0-link glycosylations of CTLA4-Igmolecules. CTLA4-Ig dimers and CTLA4-Ig single chain were treated withPNGase F to remove the N-linked oligosaccharides. The molecule was thendetected by the mass spectrometer and the corresponding ions weredeconvoluted into the spectrum. In the single chain material, thepredominant glycan composition is (HexNAc)2(Hex)2(NeuAc)2, while thereference is predominantly (HexNAc)1(Hex)1(NeuAc)1. The glycosylationcompositions are in agreement with those observed during the LC/MSpeptide analysis. In addition to a change in the O-linked glycosylati onpattern, a second major modification was observed. A mass shift of 113±4u is observed between the single chain non-reduced species and thereduced CTLA4Ig standard. The mass shift of 113±4 u disappeared uponreduction with DTT. In the dimer material, the resulting ion envelopewas deconvoluted into a spectrum (not shown herein) with a major peak at79944 amu, which corresponds to the presence of two (GalNAc)₁ (Gal)₁(NeuAc)₁ structures. The next largest peak, at 80600 amu, corresponds tothree 0-link structures or a combination of at most one branched 0-linkstructure. The third largest peak corresponds to either four O-linkedstructures or a combination containing at most two branched 0-linkstructures.

Determination of Sialic Acid Content

Another aspect of glycoprotein characterization is determination ofsialic acid. Sialic acid content can be a signature characteristic ofglycoprotein. Sialic acid content of a glycoprotein of the presentinvention can be assessed by conventional methods. For example, sialicacid can be separately determined by a direct colorimetric method (Yaoet al., 1989, Anal. Biochem., 179:332-335), using at least triplicatesamples. Another method of sialic acid determination involves the use ofthiobarbaturic acid (TBA), as described by Warren et al., 1959, J. Biol.Chem., 234:1971-1975. Yet another method involves high performancechromatography, such as described by H. K. Ogawa et al., 1993, J.Chromatography, 612:145-149.

In one embodiment, a method to determine the amount of N-AcetylNeuraminic Acid (NANA) and N-Glycolyl Neuraminic Acid (NGNA) is throughacid hydrolysis treatment of the glycoprotein of interest (for example,see Example 3). In this method, NANA and NGNA are cleaved from theprotein by acid hydrolysis. In one embodiment the glycoprotein issubstantially purifed by methods suitable for its purification. Thereleased NANA and NGNA are separated by HPLC on a Rezex MonosaccharideRHM column and detected by UV absorbance (206 nm). NANA and NGNA arequantitated based on the response factors of concurrently run NANA andNGNA standards. The results can be reported as molar ratios (MR) of NANAand NGNA respectively, to protein.

The purpose of the acid hydrolysis method of measuring sialic acidcontent is to measure the amount of total sialic acid (NANA and NGNA) toprotein in CTLA4-Ig or CTLA4^(A29YL104E)-Ig samples (molar ratios). Itis important to note, however, that these sialic acid molar ratiosinclude both bound and free NANA and NGNA. Molar ratio results areobtained based on the peak area comparison of NANA and NGNA fromhydrolyzed CTLA4-Ig or CTLA4^(A29YL104E)-Ig samples versusnon-hydrolyzed NANA and NGNA standards. Hydrolyzed standards of NANA andNGNA can also be used.

For example, molar ratios were obtained for CTLA4-Ig molecules havingSEQ ID NO:2 amino acid sequences. Without hydrolysis, the peak ofinterest in chromatograms of NANA and NGNA standards appears as a singlepeak. When the NANA standard and CTLA4-Ig samples are hydrolyzed, theresulting chromatograms show NANA as a major peak followed closely by asmall shoulder peak (<10% of the major peak area; referred to as“degraded NANA”); the same concentration of NANA standards with andwithout hydrolysis resulted in very close peak areas, including thedegradant. No peak is clearly seen in the chromatograms for a degradedNGNA species, although the area counts of the NGNA peak in a hydrolyzedNGNA standard were seen to decrease approximately 8-9%. Massspectrometry (MS) experiments demonstrated that the “NANA degradant” inboth the hydrolyzed NANA standard and the hydrolyzed CTLA4-Ig samplesresults from loss of 18 Daltons (water) from NANA. Therefore, the methodappropriately includes the small shoulder peak in the integration ofNANA peak in hydrolyzed CTLA4-Ig. It was also demonstrated by MSexperiments that NGNA degraded upon hydrolysis with a loss of 18Daltons. The NGNA degradant eluted between NANA and NANA degradant sothat UV did not detect it. In CTLA4-Ig material, NGNA content is roughly5% of NANA content and, as a result, co-elution of the NGNA degradantcauses less than 0.5% change of the NANA peak area, which is within thevariability range of the NANA peak area. The method cannot include thearea of degraded NGNA in the NGNA result; therefore the NGNA result canbe low by <10%, also within the variability of the method.

Because NGNA is thought to be more immunogenic than NANA, there is aclinical preference for a recombinant thereapeutic that contains a lowNGNA molar ratio. In one embodiment of the invention, the preponderanceof sialic acid in a population of CTLA4-Ig molecules is NANA and notNGNA, wherein in this population the molar ratio of moles sialic acidper mole CTLA4-Ig molecules or dimer is from about 5 to about 18. Inanother embodiment, the preponderance of sialic acid in a population ofCTLA4^(A29YL104E)-Ig molecules is NANA and not NGNA, wherein in thispopulation the molar ratio of moles sialic acid per moleCTLA4^(A29YL104E)-Ig molecules or dimer is from about 5.5 to about 8.5.

CTLA4-Ig and CTLA4^(A29YL104E)-Ig Expression Cassettes

The invention provides for a nucleic acid encoding a CTLA4-Ig molecule,which is an expression cassette in one embodiment. The invention alsoprovides for a nucleic acid encoding a CTLA4^(A29YL104E)-Ig molecule. Inone embodiment, the nucleic acid encoding CTLA4-Ig molecule is containedwithin an expression cassette. In another embodiment, the nucleic acidencoding the CTLA4-Ig molecule is contained within an expressioncassette derived from a plasmid having the nucleotide sequence of SEQ IDNO:17. In further embodiments, the nucleic acid encoding theCTLA4^(A29YL104E)-Ig molecule is contained within an expressioncassette. In certain embodiments, the nucleic acid encoding theCTLA4^(A29YL104E)-Ig molecule is contained within an expression cassettederived from a plasmid deposited as ATCC Accession No. PTA-2104.

The nucleic acids of the invention can be a cDNA, cDNA-like, DNA or RNAnucleic acid molecule of interest in an expressible format, such as anexpression cassette, which can be expressed from the natural promoter ora derivative thereof or an entirely heterologous promoter.Alternatively, the nucleic acid of interest can encode an anti-senseRNA. The nucleic acid of interest can encode a protein (for example aglycoprotein, such as CTLA4-Ig or CTLA4^(A29YL104E)-Ig glycoprotein),and may or may not include introns.

In one embodiment, the nucleic acid encoding a peptide having CTLA4activity can be obtained from T cell genomic DNA or from mRNA present inactivated T lymphocytes. In another embodiment, the nucleic acidencoding a CTLA4^(A29YL104E)-Ig also can be obtained from T cell genomicDNA or from mRNA present in activated T lymphocytes. In anotherembodiment of the invention, the gene encoding a protein of interest,for example CTLA4 or CTLA4^(A29YL104E)-Ig, can be cloned from either agenomic library or a cDNA according to standard protocols that oneskilled in the art practices. A cDNA, for example encoding CTLA4 orCTLA4^(A29YL104E)-Ig, can be obtained by isolating total mRNA from asuitable cell line. Using methods known in the art, double strandedcDNAs can be prepared from the total mRNA and subsequently can beinserted into a suitable bacteriophage vector or plasmid. Genes can alsobe cloned using PCR techniques well established in the art. In oneembodiment, a gene that encodes CTLA4 or CTLA4^(A29YL104E)-Ig can becloned via PCR in accordance with the nucleotide sequence informationprovided by this invention.

In another embodiment, a DNA vector containing a CTLA4 orCTLA4^(A29YL104E)-Ig cDNA can act as a template in PCR reactions whereinoligonucleotide primers designed to amplify a region of interest can beused as to obtain an isolated DNA fragment encompassing that region. Ina particular embodiment of the invention, the region of interesttargeted in CTLA4 cDNA can be the extracellular domain of CTLA4,including the extracellular domain of human CTLA4. In certainembodiments, the region of interest targeted in a CTLA4^(A29YL104E)-IgcDNA can be the extracellular domain of CTLA4 with amino acid changes atamino acid positions 55 and 130 of SEQ ID NO:2, (for example, see SEQ IDNO: 18) including the extracellular domain of human CTLA4 harboring theamino acid changes described above.

To express a fusion protein in the context of this invention, thechimeric gene fusion in one embodiment (for example a gene encoding aCTLA4-Ig immunoglobulin (CTLA4-Ig) fusion protein orCTLA4^(A29YL104E)-Ig fusion protein) includes a nucleotide sequence,which encodes a signal sequence whereby upon transcription andtranslation of the chimeric gene, directs the newly synthesized fusionprotein for secretion. In one embodiment, a native CTLA4 signal sequence(e.g., the human CTLA4 signal sequence described in Harper, K., et al.(1991, J. Immunol. 147,1037-1044) can be used. In an alternativeembodiment of the invention, a heterologous signal sequence can be usedto direct CTLA4-Ig or CTLA4^(A29YL104E)-Ig secretion (for example, theoncostatin-M signal sequence (Malik N., et al., 1989, Mol Cell Biol9(7), 2847-2853) or an immunoglobulin signal sequence). One skilled inthe art understands that the nucleotide sequence corresponding to thesignal sequence can be inserted into the chimeric gene fusion bystandard recombinant DNA techniques, such as by performing an in-frameligation of the signal sequence at the 5′ end of a nucleic acid sequenceencoding CTLA4.

Under the provisions of the Budapest Treaty, DNA encoding the amino acidsequence corresponding to a CTLA4-Ig fusion protein has been depositedwith the American Type Culture Collection (ATCC), 10801 UniversityBlvd., Manassas, Va., 20110, on May 31, 1991. It has been assigned ATCCAccession No. 68629. Additonally, an expression plasmid comprising anucleic acid sequence encoding the amino acid sequence corresponding toa CTLA4^(A29YL104E)-Ig was deposited under the provisions of theBudapest Treaty on Jun. 19, 2000 with the ATCC. The deposited plasmidhas been assigned ATCC Accession No. PTA-2104. The deposited plasmid isalso referred to as pD16 LEA29Y and pD16 L104EA29Y. CTLA4^(A29YL104E)-Igs are further described in U.S. Pat. No. 7,094,874 and co-pending U.S.Patent Application Nos. 09/579,927, 60/287,576, and 60/214,065, and inInternational Patent Publication No. WO 01/923337 A2, all of which areincorporated by reference in this application in their entireties.

An expression vector of the invention can be used to transfect cells,either eukaryotic (for example, yeast, mammalian, or insect cells) orprokaryotic in order to produce proteins (for example, fusion proteinssuch as CTLA4-Ig, CTLA4^(A29YL104E)-Ig molecules, and the like) encodedby nucleotide sequences of the vector. One skilled in the artunderstands that expression of desired protein products in prokaryotesis most often carried out in E. coli with vectors that containconstitutive or inducible promoters. Some E. coli expression vectors(also known in the art as fusion-vectors) are designed to add a numberof amino acid residues, usually to the N-terminus of the expressedrecombinant protein. Said fusion vectors can serve three functions: 1)to increase the solubility of the desired recombinant protein; 2) toincrease expression of the recombinant protein of interest; and 3) toaid in recombinant protein purification by acting as a ligand inaffinity purification. Some examples of fusion expression vectorsinclude, but are not limited to: a) pGEX (Amrad Corp., Melbourne,Australia) which fuse glutathione S-tranferase to desired protein; b)pcDNA™3.1/V5-His A B & C (Invitrogen Corp, Carlsbad, Calif.) which fuse6x-His to the recombinant proteins of interest; and c) pMAL (New EnglandBiolabs, Beverly, Mass.) which fuse maltose E binding protein to thetarget recombinant protein.

The cells suitable for culturing according to the processes and methodsof the present invention can harbor introduced expression vectors(constructs), such as plasmids and the like. The expression vectorconstructs can be introduced via transfection, lipofection,transformation, injection, electroporation, or infection. The expressionvectors can contain coding sequences, or portions thereof, encoding theproteins for expression and production in the culturing process. Suchexpression vectors can include the required components for thetranscription and translation of the inserted coding sequence.Expression vectors containing sequences encoding the produced proteinsand polypeptides, as well as the appropriate transcriptional andtranslational control elements, can be generated using methods wellknown to and practiced by those skilled in the art. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo genetic recombination which are described in J. Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols inMolecular Biology, John Wiley & Sons, New York, N.Y.

A selectable marker can be used in a recombinant expression vector (forexample, a plasmid), wherein the vector is stably integrated into thegenome of the cell, to confer resistance to the cells harboring thevector. This allows for their selection in an appropriate selectionmedium. A number of selection systems can be used, including but notlimited to, the hypoxanthine-guanine phosphoribosyltransferase (HGPRT),the Herpes Simplex Virus thymidine kinase (HSV TK), (Wigler et al.,1977, Cell, 11:223), (Szybalska and Szybalski, 1992, Proc. Natl. Acad.Sci. USA, 48:202), and adenine phosphoribosyltransferase (APRT), (Lowyet al., 1980, Cell, 22:817) genes, which can be employed in hgprt-, tk-,or aprt-cells, respectively.

The following non-limiting examples of marker genes, which can becontained within an expression vector, can also be used as the basis ofselection for anti-metabolite resistance: gpt, which confers resistanceto mycophenolic acid (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci.USA, 78:2072); dhfr, which confers resistance to methotrexate (Wigler etal., 1980, Proc. Natl. Acad. Sci. USA, 77:357; and O′Hare et al., 1981,Proc. Natl. Acad. Sci. USA, 78:1527); hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene, 30:147); and neo, which confersresistance to the aminoglycoside G418 (Clinical Pharmacy, 12:488-505; Wuand Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann. Rev.Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science, 260:926-932;Anderson, 1993, Ann. Rev. Biochem., 62:191-21; May, 1993, TIB Tech, 11(5):155-215). Recombinant DNA techniques commonly known in the art canbe routinely applied to elect the desired recombinant cell clones. Suchtechniques are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,NY; in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols inHuman Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al.,1981. J. Mol. Biol., 150:1, which are incorporated by reference hereinin their entireties.

The expression levels of an expressed protein molecule can be increasedvia amplification of the expression vector (for a review, see Bebbingtonand Hentschel, “The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning”, Vol. 3,Academic Press, New York, 1987). An increase in the level of aninhibitor present in the culture medium of a host cell will increase thenumber of copies of the marker gene when a marker in the expressionvector system expressing a protein of interest is amplifiable. Since theamplified region is associated with the protein-encoding gene, proteinproduction will concomitantly increase (Crouse et al., 1983, Mol. Cell.Biol., 3:257). Vectors that harbor the nucleic acid sequences thatencode for the selectable markers glutamine synthase (GS) ordihydrofolate reductase (DHFR) can be amplified in the presence of thedrugs methionine sulphoximine or methotrexate, respectively. Anadvantage of such vectors is the availability of cell lines, for examplethe murine myeloma cell line, NSO and the Chinese Hamster Ovary, CHO,cell line DG44, which are glutamine synthase negative and dihydrofolatereductase negative, respectively.

In one embodiment of the present invention, a nucleic acid sequenceencoding a soluble CTLA4 or CTLA4^(A29YL104E)-Ig fusion protein moleculecan be inserted into an expression vector designed for expressingforeign sequences in a eukaryotic host. The regulatory components of thevector can vary according to the eukaryotic host chosen for use. Vectorsused to express soluble CTLA4 or CTLA4^(A29YL104E)-Ig in eukaryotic hostcells can include enhancer sequences for optimization of proteinexpression.

Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like)can harbor an expression vector (for example, one that contains a geneencoding the CTLA4-Ig fusion protein or the CTLA4^(A29YL104E)-Ig fusionprotein) via introducing the expression vector into an appropriate hostcell. Accordingly, the invention encompasses expression vectorscontaining a nucleic acid sequence that encodes a CTLA4-Ig orCTLA4^(A29YL104E)-Ig fusion protein and encompasses host cells intowhich such expression vectors can be introduced via methods known in theart. As described herein, an expression vector of the invention caninclude nucleotide sequences that encode a CTLA4-Ig orCTLA4^(A29YL104E)-Ig fusion protein linked to at least one regulatorysequence in a manner that allows expression of the nucleotide sequencein a host cell. To those skilled in the art, regulatory sequences arewell known and can be selected to direct the expression of a protein ofinterest in an appropriate host cell as described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences can comprise the following:enhancers, promoters, polyadenylation signals, and other expressioncontrol elements. Practitioners in the art understand that designing anexpression vector can depend on factors, such as the choice of host cellto be transfected and/or the type and/or amount of desired protein to beexpressed.

Cloning and expression plasmids (for example, pcSD and piLN) areconstructed as described in Examples 11. In one embodiment of thisinvention, an isolated DNA fragment from plasmid pSV2dhfr-is ligated tothe pcDNA3 vector backbone generating the expression vector pcSD. VectorpcSD is comprised of the following features: a cytomegalovirus (CMV)promoter followed by a multiple cloning site (MCS); a bovine growthhormone (BGH) polyadenylation signal and transcriptional terminationsequence; a mouse dhfr cDNA sequence for selection and amplification; anampicillin resistance gene; and a pUC origin of replication forselection and maintenance in Escherichia coli. Vector piLN isconstructed containing cDNAs encoding portions of the amino acidsequence corresponding to a fragment of the extracellular domain of thehuman CTLA4 receptor Example 11, where the cDNA encoding a first aminoacid sequence is joined to DNA encoding a second amino acid sequence,which corresponds to an IgC region that permits the expression of theCTLA4 receptor gene by altering the solubility of the expressed CTLA4protein (see FIG. 1 and brief description for FIG. 1 for residuescorresponding to CTLA4 extracellular portion and IgGl constant region).In one embodiment, an oncostatin M signal peptide sequence can be fusedto the amino acid sequence corresponding to the extracellular domain ofCTLA4 which subsequently is fused to a second amino acid sequencecorresponding to an Ig domain (for example, the human IgC_(γ1) domain)as previously described in FIG. 1. The oncostatin M signal sequenceallows for soluble forms of the CTLA4 gene (for example CTLA4-Ig)protein product to be generated.

To construct a pcSD expression vector containing a gene encoding theCTLA4-immunoglobulin fusion protein, methods known in the art (forexample, restriction site sub-cloning) can be used. The startingmaterial for one embodiment of the invention can be a digested andexcised DNA fragment from the cloning vector piLN described in Example11. In another embodiment, the excised DNA fragment from said vectorcontains the amino acid sequence of the oncostatin M signal sequence andCTL4Ig fusion protein, wherein said DNA fragment is ligated to thedigested pcSD vector. The oncostatin M-CTLA4-Ig DNA fragment can beinserted between the CMV promoter and a cassette containing the BGHpolyadenylation signal and transcriptional termination sequence. Thiswould place a CTLA4-Ig gene product under the control of the CMVpromoter in the plasmid designated pcSDhuCTLA4-Ig (FIG. 20; SEQ ID NO:17).

Additionally, cloning and expression plasmids (for example, pD16 LEA29Y)can be derived from the Invitrogen plasmid pcDNA3. Vector pD16 LEA29Y(FIG. 21) comprises the following features: the neomycin resistance genefrom pcDNA3 was replaced with the murine dihydrofolate reductase (DHFR)gene under control of the enhancerless (weakened) SV40 promoter; thegene encoding a CTLA4^(A29YL104E)-Ig is expressed from the CMV promoter,and the poly adenylation signal is from the bovine growth hormone gene;the expression cassette for the gene of interest is flanked bytranscription termination sequences, i.e., 5′ to the promoter and 3′ tothe poly A site; the vectors contain two distinct restriction sitepolylinkers, one 3′ to the promoter for cloning the gene of interest,and one 5′ to the promoter for vector linearization prior totransfection; the vector contains an ampicillin resistance gene and theColE1 origin of replication for plasmid propagation in E. coli; theCTLA4^(A29YL104E)-Ig sequence (SEQ ID NO:3) is preceded by theOncostatin M signal peptide and assembled in the expression vector knownas vector pD16 LEA29Y.

The vector is constructed containing cDNAs encoding portions of theamino acid sequence corresponding to a fragment of the extracellulardomain of the human CTLA4 receptor (SEQ ID NO:2), wherein the amino acidAla at position 55 is replaced by the amino acid Tyr and the amino acidLeu at position 130 is replaced by the amino acid Glu (FIG. 3). Theseamino acid changes are depicted in the CTLA4^(A29YL104E)-Ig amino acidsequence having SEQ ID NO: 4. The cDNA encoding a first amino acidsequence (for example, the sequence that encodes a CTLA4^(A29YL104E)-Ig)is joined to DNA encoding a second amino acid sequence, whichcorresponds to an IgC region that permits the expression of theCTLA4^(A29YL104E)-Ig receptor gene by altering the solubility of theexpressed CTLA4^(A29YL104E)-Ig protein (see FIG. 3 and the briefdescription for FIG. 3 for residues corresponding to the modified CTLA4extracellular portion and IgG1 constant region) having SEQ ID NO: 3.

In one embodiment, an oncostatin M signal peptide sequence can be fusedto the amino acid sequence corresponding to the extracellular domain ofCTLA4 which subsequently is fused to a second amino acid sequencecorresponding to an Ig domain (for example, the human IgC_(yi) domain)as previously described in FIG. 3. The oncostatin M signal sequenceallows for soluble forms of the CTLA4 gene (for example aCTLA4^(A29YL104E)-Ig) protein product to be generated.

Stable Transfection to Generate Cell Line

Vectors that contain DNA encoding a protein of interest (for example,fusion constructs, glycoproteins, and the like) can be transformed intosuitable host cells (for example bacterial cells) in order to producelarge quantities of cloned DNA. Some non-limiting examples of bacterialcells for transformation include the bacterial cell line E. coli strainsDH5α or MC1061/p3 (Invitrogen Corp., San Diego, Calif.), which can betransformed using standard procedures practiced in the art, and coloniescan then be screened for the appropriate plasmid expression.

Expression vectors for eukaryotic cells, such as mammalian cells, caninclude promoters and control sequences compatible with mammalian cells.In one embodiment of the invention, these regulatory elements can be,for example, a CMV promoter found in the pcSD or pD16 LEA29Y vector, orthe avian sarcoma virus (ASV) located in the piLN vector. Other commonlyused early and late promoters include, but are not limited to, thosefrom Simian Virus 40 (SV 40) (Fiers, et al., 1973, Nature 273:113), orother viral promoters such as those derived from bovine papilloma,polyoma, and Adenovirus 2 virus. The regulatable promoter, hMTII (Karin,et al., 1982, Nature 299:797-802) can also be used, in addition toothers known in the art. For recombinant protein expression in culturedinsect cells (for example, SF 9 cells), some baculovirus vectorsavailable include the pVL series (Lucklow, V. A., and Summers, M. D.,1989, Virology 170:31-39) and the pAc series (Smith et al., 1983, Mol.Cell Biol. 3:2156-2165). A practitioner skilled in the art alsounderstands that enhancer regions (those sequences found upstream ordownstream of the promoter region in non-coding DNA regions) are alsoimportant in optimizing expression. Origins of replication can beemployed, if needed, from viral sources, for example if utilizing aprokaryotic host for introduction of plasmid DNA. However, chromosomeintegration is a common mechanism for DNA replication in eukaryoticorganisms.

Although in an embodiment of this invention mammalian host cells (suchas CHO cells) are employed for expression of desired protein (forexample, fusion proteins, glycoproteins, and the like), other eukaryoticorganisms also may be used as hosts. Laboratory strains of the buddingyeast Saccharomyces cerevisiae (also known as Baker's yeast or Brewer'syeast) can be used as well other yeast strains, such as the fissionyeast Schizosaccharomyces pombe. Yeast vectors harboring DNA encoding aprotein of interest (for example fusion constructs, glycoproteins, andthe like such as CTLA4-Ig or CTLA4^(A29YL104E)-Ig), can utilize the 2 μorigin of replication of Broach, Meth. Enz. 101:307 (1983), or otherorigins of replications compatible with yeast (for example, Stinchcombet al., 1979, Nature 282:39; Tschempe et al., 1980, Gene 10:157; andClarke et al., 1983, Meth. Enz. 101:300). A regulatory element containedwithin yeast vectors can be a promoter for the synthesis of glycolyticenzymes (Hess et al., 1968, J. Adv. Enzyme Reg. 7:149; Holland et al.,1978, Biochemistry 17:4900).

One skilled in the art can also utilize other promoters wherein growthconditions can regulate transcription of said regulatable gene, and caninclude the following non-limiting examples: isocytochrome C, alcoholdehydrogenase 2, enzymes responsible for maltose and galactoseutilization, acid phosphatase, and degradative enzymes associated withnitrogen metabolism. Similar to mammalian expression systems, terminatorsequences in yeast expression vectors are also desirable at the 3′ endof the coding sequences and are found in the 3′ untranslated regionfollowing the open reading frame in yeast-derived genes. Somenon-limiting examples of yeast vectors suitable for recombinant proteinexpression in yeast (for example, in S. cerevisiae) include pMFa (Kurjanand Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., 1987,Gene 54:113-123), pYepSecl (Baldari, et al., 1987, Embo J. 6:229-234),pYES2 (Invitrogen Corporation, San Diego, Calif.), as well as thosebelonging to the pRS family of yeast vectors.

Clones, for example bacterial clones, which contain DNA encoding aprotein of interest (for example, fusion constructs, glycoproteins, andthe like), obtained as described above may then transfected intosuitable host cells, such as mammalian cells, for expression of thedesired product. Transfection techniques are carried out using standardtechniques established in the art appropriate to said host cells,wherein the transfection technique depends on the host cell used. Forexample, mammalian cell transfection can be accomplished usinglipofection, protoplast fusion, DEAE-dextran mediated transfection,CaPO₄ co-precipitation, electroporation, direct microinjection, as wellas other methods known in the art which can comprise: scraping, directuptake, osmotic or sucrose shock, lysozyme fusion or erythrocyte fusion,indirect microinjection such as via erythrocyte-mediated techniques,and/or by subjecting host cells to electric currents. As othertechniques for introducing genetic information into host cells will bedeveloped, the above-mentioned list of transfection methods is notconsidered to be exhaustive.

Expression of DNA encoding a protein of interest (for example, fusionconstructs, glycoproteins, and the like) in eukaryotic host cellsderived from multicellular organisms (for example, mammalian in origin)is particularly utilized in the context of this invention (TissueCultures, Academic Press, Cruz and Patterson, Eds. (1973)). Host cellsderived from multicellular organisms have the ability to splice outintrons and thus can be used directly to express genomic DNA fragments.As stated earlier, useful host cell lines include, but are not limitedto, Chinese hamster ovary (CHO), BHK cells, monkey kidney (COS), VEROand HeLa cells. In the present invention, cell lines stably expressingthe protein of interest (for example, fusion constructs, glycoproteins,and the like) are used. In one embodiment, a mammalian cell line, (suchas a CHO cell line) is transfected (for example by electroporation) withan expression vector (for example, pcSDhuCTLA4-Ig, pD16 LEA29Y, and thelike) containing a DNA sequence encoding a glycoprotein of interest. Inone embodiment, the glycoprotein of interest can be a CTLA4-Ig protein,including the CTLA4-Ig protein(s) having an amino acid sequencecontained in SEQ ID NO:2, encoded by a portion of the nucleotidesequence in SEQ ID NO:1. In another embodiment, the glycoprotein ofinterest can be a CTLA4^(A29YL104E)-Ig, including theCTLA4^(A29YL104E)-Ig having an amino acid sequence contained in SEQ IDNO:4, encoded by a portion of the nucleotide sequence in SEQ ID NO:23.

A recombinant protein, such as CTLA4-Ig or CTLA4^(A29YL104E)-Ig, can beexpressed in eukaryotic host cells, such as mammalian cells (forexample, CHO, BHK, VERO, or NSO cells), insect cells (for example, usinga baculovirus vector), or yeast cells. Those skilled in the art can useother suitable host cells, such as those described earlier, in thecontext of this invention. In one embodiment, eukaryotic, rather thanprokaryotic, expression of a recombinant fusion protein, (such asCTLA4-Ig or CTLA4^(A29YL104E)-Ig) is employed. Expression of eukaryoticrecombinant proteins, such as human CTLA4-Ig or CTLA4^(A29YL104E)-Ig, ineukaryotic cells, such as CHO cells, can lead to partial and/or completeglycosylation, as well as the formation of intra-or inter-chaindisulfide bonds. For transient amplification and expression of a desiredprotein, a vector harboring DNA encoding a protein of interest (forexample fusion constructs, glycoproteins, and the like such as CTLA4-Igor CTLA4^(A29YL104E)-Ig), is delivered into eukaryotic cells by atransfection method known in the art but not integrated into the cell'sgenome. Expression of transfected genes can be measured within 16-96hours. Mammalian cells (such as COS cells) can be used in conjunctionwith vectors such as pCDM8 to transiently express a desired protein(Gluzman, Y., 1981, Cell 23:175-182; Seed, B., 1987, Nature 329:840).

It is understood in the art that for stable transfection of mammaliancells, a small fraction of cells can integrate DNA into their genomesand successful integration can depend on the expression vector andtransfection method utilized. For stable amplification and expression ofa desired protein, a vector harboring DNA encoding a protein of interest(for example fusion constructs, glycoproteins, and the like such asCTLA4-Ig or CTLA4^(A29YL104E)-Ig) is stably integrated into the genomeof eukaryotic cells (such as mammalian cells), resulting in the stableexpression of transfected genes. In order to identify and select clonesstably expressing a gene that encodes a protein of interest, a gene thatencodes a selectable marker (for example, resistance to antibiotics) canbe introduced into the host cells along with the gene of interest.Selectable markers used by one skilled in the art can be those thatconfer resistance to drugs, such as G418 and hygromycin. The geneencoding a selectable marker can be introduced into a host cell on aseparate plasmid or can be introduced on the same plasmid as the gene ofinterest. Cells containing the gene of interest can be identified bydrug selection wherein cells that have incorporated the selectablemarker gene will survive in the presence of said drug, while cells thathave not incorporated the selectable marker gene die. Surviving cellscan then be screened for the production of the desired protein (forexample, a CTLA4-Ig protein or CTLA4^(A29YL104E)-Ig).

As described earlier, CHO cells deficient in expression of thedihydrofolate reductase (dhfr) gene can survive only with the additionof nucleosides. When said cells are stably transfected with a DNA vectorharboring the dhfr gene, cells are then capable of producing thenecessary nucleosides. By using dhfr as the selectable marker, oneskilled in the art understands that in the presence of theanti-metabolite, methotrexate, gene amplification of dhfr as well as thetransfected gene of interest (for example, CTLA4-Ig orCTLA4^(A29YL104E)-Ig) readily occurs. In one embodiment of thisinvention, mammalian cells, such as CHO dhfr-cells, are transfected withan expression vector, such as pcSDhuCTLA4-Ig (Examples 11-13) or pD16LEA29Y, to generate a population of cells that can be stably amplifiedand that can stably express a desired protein product, (such as CTLA4-Igor beta p CTLA4^(A29YL104E)-Ig olypeptide, respectively). In anotherembodiment, the dhfr-negative cell line DG44 (Invitrogen Corp. Carlsbad,Calif.) can be employed for stable transfection. In another embodimentof this invention, transfection can occur via electroporation.

As is readily practiced in the art, transfected mammalian cells (forexample dhfr-negative CHO cells) are maintained in non-selective mediumcontaining serum for 1-2 days post-transfection. Cells then are treatedwith trypsin and re-plated in serum-containing medium, in the presenceof a selective pressure (for example, a drug such as methotrexate).Cells are cultured in selective serum-containing medium for 2-3 weeks,with frequent changes of selective medium in order to eliminate debrisand dead cells, until distinct colonies can be visualized. Individualcolonies can then be trypsinized and placed into multi-well plates forfurther propagation and amplification in the presence of selectivemedium in order to identify producers that express a high level of thedesired protein (for example, fusion constructs, glycoproteins, and thelike) via methods established in the art such as ELISAs orimmunoprecipitation. In one embodiment of this invention, the methoddescribed above was carried out for transfecting dhfr-negative CHO cells(for example DG44 cells) in order to establish a stable cell lineexpressing a recombinant protein of interest (for example, a CTLA4-Igprotein) (see for example, Examples 12-13). In another embodiment, astable cell line expressing a CTLA4^(A29YL104E)-Ig was established (seeEXAMPLE 23).

A stable CHO line of the invention stably expresses CTLA4-Ig proteinmolecules as CTLA4-Ig monomers having the sequence (i) 26-383 of SEQ IDNO:2, (ii) 26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv)27-382 of SEQ ID NO:2, (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQID NO:2. This cell line can secrete a population of CTLA4-Ig moleculesthat can exist as multimeric forms (such as dimers, tetramers, and thelike), wherein the multimeric form can have different monomer sequencesof SEQ ID NO:2. The expression cassette integrated into this cell linecomprises SEQ ID NO:1, and is contained within pcSDhuCTLA4-Ig.

The invention also provides a stable CHO line, which stably expresses aCTLA4^(A29YL104E)-Ig. In one embodiment, the cell line expressesCTLA4^(A29YL104E)-Ig monomers having the sequence (i) 26-383 of SEQ IDNO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQ ID NO:4, (iv)27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi) 25-383 of SEQID NO:4. In another embodiment, the cell line can secrete a populationof CTLA4^(A29YL104E)-Ig molecules that can exist as multimeric forms(such as dimers, tetramers, and the like), wherein the multimeric formcan have different monomer sequences of SEQ ID NO:4. The expressioncassette integrated into this cell line comprises SEQ ID NO:3, and iscontained within pD16 LEA29Y.

Subcloning To Generate a Clonal Population of Cells

Cells identified as being producers of a desired protein (for example,fusion constructs, glycoproteins, and the like) are isolated from cellculture and subsequently amplified under production-equivalentconditions wherein culture medium can contain serum. Subcloning methodsknown in the art, such as, but not limited to, soft-agar cloning, can beemployed. The stable recombinant cell clones obtained can then befurther multiplied under serum-and animal product-free conditions.According to the present invention, a stable cell clone expressing thedesired protein product (for example CTLA4-Ig, a CTLA4^(A29YL104E)-Ig,and the like) is achieved via obtaining a recombinant cell clone from acell culture that is obtained after culturing a recombinant originalcell clone in serum-containing medium and re-adapting the cells toserum-and animal product-free medium. In one embodiment, the cell clonesexpressing CTLA4-Ig can be continued to be cultured in serum-and animalproduct-free medium through at least 50 generations. In anotherembodiment of the invention, the cell clones can be continued to becultured as described above through at least 75 generations. Accordingto the invention, cell clones can also be continued to be cultured inserum-and animal product-free medium through at least 100 generations.

In a further embodiment, the cell clones expressing aCTLA4^(A29YL104E)-Ig can be continued to be cultured in serum-and animalproduct-free medium through at least 27 generations. In anotherembodiment, the cell clones can continue to be cultured as describedabove through at least 75 generations. Additionally, cell clones can becultured in serum-and animal product-free medium through at least 100generations.

In one embodiment, the invention provides a cell line that producesCTLA4-Ig molecules comprising SEQ ID NO:2 monomers, wherein the cellline is stable for over 100 generations, and wherein cell line stabilitycomprises: (1) doubling time at generation 100 is less than about24.5±2.6 hours; (2) cell viability at generation 100 is greater than95%, (3) production titer for CTLA4-Ig in 5-L bioreactors is greaterthan 1.69 mg/mL at generation 100; (4) sialic acid molar ratio toprotein is about 9.3 to about 11.0 at generation 105.

The stable recombinant cell clone of this invention is present inisolated form wherein isolation can occur according to methods practicedin the art (for example, soft-agar cloning or limited dilution cloningor the like). In this invention, the stable recombinant cell clone isderived from a recombinant mammalian cell (for example, a CHO cell) thatcontains DNA sequences encoding a recombinant protein of interest (forexample, fusion constructs, glycoproteins, and the like, such asCTLA4-Ig or CTLA4^(A29YL104E-)Ig), which can grow in suspension oradherently. A recombinant protein expressed by the cell line of thisinvention can be a therapeutic glycoprotein, such as CTLA4-Ig orCTLA4^(A29YL104E)-Ig. According to the present invention, stablerecombinant cell clones derived from eukaryotic cells (such as mammalianCHO cells, DG44 cells, or dhfr-negative CHO cells), which contain a DNAsequence encoding a recombinant glycoprotein, such as CTLA4-Ig orCTLA4^(A29YL104E)-Ig, and which are capable of stably expressing therecombinant glycoprotein over several generations is useful.

In one embodiment of the invention, a population of mammalian host cellsstably expressing a protein of interest (for example, fusion constructs,glycoproteins, and the like, such as CTLA4-Ig or CTLA4^(A29YL104E)-Ig)is obtained under serum-and animal product-free conditions viaamplifying the stably transfected cells. According to the invention, arecombinant cell clone can then be characterized in that it is stable inserum-free and animal product-free culturing medium through at least 105generations, for example.

In one embodiment of the invention, the clonal population of cellsproduces CTLA4-Ig molecules. Some of the specific characteristics ofthis population of CTLA4-Ig molecules are listed below in Table 6. Apopulation of CTLA4-Ig molecules can at least includes CTLA4-Ig dimermolecules that comprise two monomer molecules that each can have one ofthe following sequences: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQID NO:2, (iii) 27-383 of SEQ ID NO:2, (iv) 27-382 of SEQ ID NO:2, (v)25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2. Thus, thepopulation of CTLA4-Ig molecules can include predominantly homodimers orheterodimers. The population can include both homodimers andheterodimers. In one embodiment, the invention provides for a populationof CTLA4-Ig molecules having the characteristics shown in Table 6 or apharmaceutical equivalent thereof. As used herein, a pharmaceuticalequivalent is where a population of molecules has a safety and efficacyprofile equivalent to the original population (standard population) fortreating a patient, as would be understood by a governmental agency,such as the FDA. For example, the CTLA4-Ig population of this inventioncan have the characteristics shown in Table 6. In another embodiment,the population of CTLA4-Ig molecules of the invention can have thecharacteristics shown in Table 6 or equivalents thereof singly or in anycombination or permutaion thereof

In another embodiment, the clone of interest can also be characterizedaccording to the recombinant product expressed and its biochemicalcharacteristics (for example, CTLA4-Ig having a particular extinctioncoefficient value). An extinction coefficient value (also referred to asan absorptivity value (a_(s))) can be derived theoretically orexperimentally. At 280 nm, the absorptivity value (a_(s)) of CTLA4-Igwas determined to be 1.01 mL mg⁻¹ cm⁻¹ using the method of Mach, et al.(Analytical Biochemistry, Vol. 200, pp. 74-80, 1992) as detailed below.Equation 1 was used to determine the molar absorptivity (ε).

ε=[(Number of disulfide bonds×134)+(Number of Tryptophanresidues×5,540)+(Number of Tyrosine residues×1,480)]  Equation 1

CTLA4-Ig has 9 disulfide bonds, 8 tryptophan residues and 32 tyrosineresidues to give a molar absorptivity (c) of 92,886 M⁻¹cm⁻¹ as shown inEquation 2.

ε=(9×134)+(8×5,540)+(32×1,480) ]=92,886 M⁻¹ cm⁻¹   Equation 2

The absorptivity constant (a_(s)) was calculated by dividing the molarabsorptivity (ε) by the molecular weight where the molecular weight wasdetermined by MALDI-TOF as shown in Equation 3:

a _(s)=ε/Molecular Weight=92,886 M⁻¹ cm⁻¹/92,278 Da=1.01 mL mg⁻¹ cm⁻¹  Equation 3

A comparison of the theoretically derived absorptivity value to theexperimentally determined absorptivity values on two lots of CTLA4-Ig(comprising SEQ ID NO:2) material was carried out using amino acidanalysis. The average experimentally determined absorptivity constant is1.00±0.05 mL mg⁻¹ cm⁻¹. The experimental value confirms the theoreticalvalue of 1.01 mL mg⁻¹ cm⁻¹ within the error of the experimentaldetermination. Thus, in one embodiment, the invention provides a cellline that produces CTLA4-Ig molecules that have an absorptivity value orextinction coefficient of about 1.00±0.05 mL mg⁻¹ cm⁻¹.

According to this invention, the recombinant clone of interest can alsobe characterized according to the number of sites a DNA sequence thatencodes a protein of interest (for example, fusion constructs,glycoproteins, and the like, such as CTLA4-Ig) is integrated into thehost cell genome. One skilled in the art understands that standardSouthern hybridization techniques will allow for such an analysis. Inone embodiment of the invention, a single hybridizing fragment ofapproximately 1.2 kb was detected in each of the EcoRI, and XbaIrestriction digests of genomic DNA prepared from the recombinant cellclone of the invention, consistent with the expected size of theCTLA4-Ig gene (Southern hybrid; FIG. 22). The figure depiction isconsistent with a single integration site of the plasmid as well asthere being no insertions or deletions in the CTLA4-Ig gene beingdetectable by Southern hybridization analysis.

In one embodiment, the invention provides CHO cell populations capableof producing CTLA4-Ig molecules, wherein each cell of the populationcomprises at least 30 copies of a nucleic acid that codes for a CTLA4-Igprotein, wherein the 30 or more copies are integrated in tandem at asingle site in the genome of the cell, and wherein the population ofcells are clonal. In other embodiments, the CHO cell populations capableof producing CTLA4-Ig molecules comprise a population wherein at least75%, 80%, 85%, 90%, 95%, or 99% of the cells in the populationscomprises at least 30 copies of a nucleic acid that codes for a CTLA4-Igprotein.

In another embodiments, the invention provides a cell line that whencultured in conditions according to Example 14, produces CTLA4-Igmolecules comprised of SEQ ID NO:2 in an amount that is least 1.0, 1.3,1.6, 1.8, 2.0, or 2.5 grams of CTLA4-Ig molecules per liter cell cultureat the production stage.

According to the invention, a mammalian cell line (for example a dhfrnegative CHO cell line) is generated which expresses a desired protein(for example a CTLA4-Ig protein) that when grown in a suspended culturecan produce a population of molecules that is secreted into the culturesupernatant. This population of molecules can have, for example, one ormore or all of the following characteristics listed in TABLE 6.

TABLE 6 Illustrative CTLA4-Ig Characteristics Characteristic 1N-terminal Sequence aa 26 (Ala) of SEQ ID NO: 2 aa 27 (Met) of SEQ IDNO: 2 2 C-terminal Sequence aa 382 (Gly) of SEQ ID NO: 2 aa 383 (Lys) ofSEQ ID NO: 2 3 B7 Binding 70-130% 4 pI 4.3-5.6 5 Sialic Acid Ratio ≥8.0moles per mole CTLA4-Ig molecules NANA 8.0-12.0 moles per mole CTLA4-Igmolecules NGNA ≤1.5 moles per mole CTLA4-Ig molecules 6 dimer ≥95% 7 HMWSpecies (e.g. ≤4.0% tetramer) 8 Low Molecular Weight ≥0.5% Species (e.g.monomer) 9 Exctinction Coefficient 1.0 ± 0.05 ml/mg · cm 10 FreeSulfhydryl Groups ≤0.24 free thiols per molecule 11 Amino Monosaccharide15-35 moles per mole Composition: GlcNAc CTLA4-Ig molecules 12 AminoMonosaccharide 1.7-8.3 moles per mole Composition: GalNAc CTLA4-Igmolecules 13 Neutral Monosaccharide 8-17 moles per mole Composition:Galactose CTLA4-Ig molecules 14 Neutral Monosaccharide 3.5-8.3 moles permole Composition: Fucose CTLA4-Ig molecules 15 Neutral Monosaccharide7.7-22 moles per mole Composition: Mannose CTLA4-Ig molecules

According to Table 6, the percent of CTLA4-Ig dimer, percent of HMWspecies (for example CTLA4-Ig multimers such as a tetramer), and percentof LMW species (for example CTLA4-Ig monomer) are with respect to apopulation of CTLA4-Ig molecules. The moles of sugars and the moles ofsialic acid described in Table 6 are with respect to mole of CTLA4-Igmolecules or dimer. The percent of B7 binding found in Table 6 is inreference to CTLA4-Ig binding experiments performed by surface plasmonresonance (SPR) on a BIAcore instrument described earlier wherein thepercentage is a comparison to B7 binding to a CTLA4-Ig control.

In one embodiment, a mammalian cell line (such as, a dhfr negative CHOcell line) generates a population of CTLA4-Ig molecules displayingcharacteristic attributes numbers 1-5 from Table 6. In anotherembodiment of the invention, a mammalian cell line generates apopulation of CTLA4-Ig molecules having characteristic attributesnumbers 1-10 from Table 6. In other embodiments, a mammalian cell linegenerates a population of CTLA4-Ig molecules displaying characteristicattributes numbers 1-15 from Table 6. In a further embodiment, theamount of free sulfhydryl groups on CTLA4-Ig is about ≤0.20 free thiolsper molecule.

Upon purification of the cell culture supernatant that contains thedesired protein (for example a CTLA4-Ig protein) secreted by apopulation of transfected mammalian cells (for example a dhfr negativeCHO cell), the population of molecules can have further characteristics.In addition to those characteristics listed in Table 6, this populationof molecules can have, for example, one or more or all of the followingcharacteristics: a pH range from about 7.0-8.0; ability to inhibit humancell IL-2 activity by 50-150%; Monocyte Chemotactic Protein (MCP-1)present in the final purified product at ≤5 ng/mg CTLA4-Ig dimer orCTLA4-Ig molecules; concentration of DNA present in the final purifiedproduct at ≤2.5 pg/mg CTLA4-Ig dimer; CHO host cell protein present inthe final purified product at ≤50 ng/mg CTLA4-Ig dimer; concentration ofTriton X-100 in the final purified product at ≤1.0 ppm; amount ofProtein A at ≤5 ng/mg CTLA4-Ig dimer; amount of bacterial endotoxins inthe final purified product at ≤0.3 EU/mg CTLA4-Ig dimer; amount ofBioburden in the final purified product at ≤3.0 CFU/10 ml.

In one embodiment, Monocyte Chemotactic Protein (MCP-1) is present inthe final purified product at ≤3 ng/mg CTLA4-Ig dimer or CTLA4-Igmolecules; the concentration of DNA present in the final purifiedproduct at ≤1.0 pg/mg CTLA4-Ig dimer; CHO host cell protein present inthe final purified product at ≤10 ng/mg CTLA4-Ig dimer; amount ofProtein A at lng/mg CTLA4-Ig dimer; amount of bacterial endotoxins inthe final purified product at 0.15 EU/mg CTLA4-Ig dimer; and amount ofBioburden in the final purified product at 1.0 CFU/10m1; a pH range fromabout 7.2-7.8. In a particular embodiment, Monocyte Chemotactic Protein(MCP-1) is present in the final purified product at ≤1 ng/mg CTLA4-Igdimer or CTLA4-Ig molecules. In a further embodiment, CTLA4-Ig moleculesinhibit human cell IL-2 activity by 60-140%.

In another embodiment of the invention, the clonal population of cellsproduces CTLA4^(A29YL104E)-Ig molecules. Some of the specificcharacteristics of this population of CTLA4^(A29YL104E)-Ig molecules arelisted in Table 7. A population of CTLA4^(A29YL104E)-Ig molecules can atleast includes CTLA4^(A29YL104E)-Ig dimer molecules that comprise twomonomer molecules that each can have one of the following sequences: (i)26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4, (iii) 27-383 of SEQID NO:4, (iv) 27-382 of SEQ ID NO:4, (v) 25-382 of SEQ ID NO:4, and (vi)25-383 of SEQ ID NO:4. Thus, the population of CTLA4^(A29YL104E)-Igmolecules can include predominantly homodimers or heterodimers, or anymixture thereof. In one embodiment, the invention provides for apopulation of CTLA4^(A29YL104E)-Ig molecules having the characteristicsshown in Table 7 or a pharmaceutical equivalent thereof. As used herein,a pharmaceutical equivalent is where a population of molecules has asafety and efficacy profile equivalent to the original population(standard population) for treating a patient, as would be understood bya governmental agency, such as the FDA. For example, theCTLA4^(A29YL104E)-Ig population of this invention can have thecharacteristics shown in Table 7. In another embodiment, the populationof CTLA4^(A29YL104E)-Ig molecules of the invention can have thecharacteristics shown in Table 7 or equivalents thereof singly or in anycombination or permutation thereof.

In one embodiment, the invention provides CHO cell populations capableof producing CTLA4^(A29YL104E)-Ig molecules, wherein each cell of thepopulation comprises at least 30 copies of a nucleic acid that codes fora CTLA4^(A29YL104E)-Ig protein, wherein the 30 or more copies areintegrated in tandem at a single site in the genome of the cell, andwherein the population of cells are clonal. In other embodiments, theCHO cell populations capable of producing CTLA4^(A29YL104E)-Ig moleculescomprise a population wherein at least 75%, 80%, 85%, 90%, 95%, or 99%of the cells in the populations comprises at least 30 copies of anucleic acid that codes for a CTLA4^(A29YL104E)-Ig.

In another embodiments, the invention provides a cell line that whencultured in conditions according to FIG. 23 or Examples 19-20, producesCTLA4^(A29YL104E)-Ig molecules comprised of SEQ ID NO:4 in an amountthat is least 22, 22.5, 23, 27.5, or 28 grams of CTLA4^(A29YL104E)-Igmolecules per liter cell culture at the production stage.

According to the invention, a mammalian cell line (for example a dhfrnegative CHO cell line) is generated which expresses a desired protein(for example a CTLA4^(A29YL104E)-Ig) that when grown in a suspendedculture can produce a population of molecules that is secreted into theculture supernatant. This population of molecules can have, for example,one or more or all of the following characteristics listed in TABLE 7.

TABLE 7 Illustrative Characteristics of a CTLA4^(A29YL104E)-IgCharacteristic 1 N-terminal Sequence aa 26 (Ala) of SEQ ID NO: 4 aa 27(Met) of SEQ ID NO: 4 2 C-terminal Sequence aa 382 (Gly) of SEQ ID NO: 4aa 383 (Lys) of SEQ ID NO: 4 3 B7 Binding 70-130% 4 pI 4.5-5.5   5Sialic Acid Ratio ≥5.0 moles per mole Total CTLA4-Ig protein 6 Dimer≥95%  7 HMW Species (e.g., ≤4% tetramer) 8 LMW species (e.g., ≤1%monomer) 9 Amino Monosaccharide 24-28 moles per mole Total Composition:GlcNAc CTLA4-Ig protein 10 Amino Monosaccharide 2.7-3.6 moles per moleTotal Composition: GalNAc CTLA4-Ig protein 11 Neutral Monosaccharide11-13 moles per mole Total Composition: Galactose CTLA4-Ig protein 12Neutral Monosaccharide 6.4-7.0 moles per mole Total Composition: FucoseCTLA4-Ig protein 13 Neutral Monosaccharide 14-16 moles per mole TotalComposition: Mannose CTLA4-Ig protein

According to Table 7, the percent of CTLA4^(A29YL104E)-Ig dimer, percentof HMW species (for example CTLA4^(A29YL104E)-Ig multimers such as atetramer), and percent of LMW species (for example CTLA4^(A29YL104E)-Igmonomer) are with respect to a population of CTLA4^(A29YL104E)-Igmolecules. The moles of sugars and the moles of sialic acid described inTable 7 are with respect to mole of CTLA4^(A29YL104E)-Ig molecules ordimer. The percent of B7 binding found in Table 1 is in reference toCTLA4^(A29YL104E)-Ig binding experiments performed by surface plasmonresonance (SPR) on a BIAcore instrument described earlier wherein thepercentage is a comparison to B7 binding to a CTLA4^(A29YL104E)-Igcontrol.

In one embodiment, a mammalian cell line (such as, a dhfr negative CHOcell line) generates a population of CTLA4^(A29YL104E)-Ig moleculesdisplaying characteristic attributes numbers 1-5 from Table 7. Inanother embodiment of the invention, a mammalian cell line generates apopulation of CTLA4^(A29YL104E)-Ig molecules having characteristicattributes numbers 1-10 from Table 7. In other embodiments, a mammaliancell line generates a population of CTLA4^(A29YL104E)-Ig moleculesdisplaying characteristic attributes numbers 1-13 from Table 7.

Upon purification of the cell culture supernatant that contains thedesired protein (for example a CTLA4^(A29YL104E)-Ig) secreted by apopulation of transfected mammalian cells (for example a dhfr negativeCHO cell), the population of molecules can have further characteristics.In addition to those characteristics listed in Table 7, this populationof molecules can have, for example, one or more or all of the followingcharacteristics: Monocyte Chemotactic Protein (MCP-1) present in thefinal purified product at ≤5 ng/mg CTLA4^(A29YL104E)-Ig dimer;concentration of DNA present in the final purified product at ≤2.5 pg/mgCTLA4^(A29YL104E)-Ig dimer; and CHO host cell protein present in thefinal purified product at ≤50 ng/mg CTLA4^(A29YL104E)-Ig dimer.

General Culturing of Cell Lines

According to this invention, mammalian cells are cultured to produce adesired protein, including a glycoprotein, as conventionally known byone skilled in the art. The mammalian cells expressing a glycoprotein ofinterest should express or be manipulated to express the appropriateenzymes such that under satisfactory conditions, post-translationalmodifications most pertinent to glycosylation occur in vivo. The enzymesinclude those necessary for the addition and completion of N-andO-linked carbohydrates, such as those described in Hubbard and Ivatt,Ann. Rev. Biochem., 50:555-583(1981) for N-linked oligosaccharides. Theenzymes optionally include oligosaccharyltransferase, alpha-glucosidaseI, alpha-glucosidase II, ER alpha(1,2)mannosidase, Golgi alpha-mannodaseI, N-acetylyglucosaminyltransferase I, Golgi alpha-mannodase II,N-acetylyglucosaminyltransferase II, alpha(1,6)fucosyltransferase, beta(1,4)galactosyltransferase, and an appropriate sialyltransferase.

A delay in apoptosis (programmed cell death) can have an effect ofincreasing cell viability during a cell culturing processes. A decreasein apoptosis, and in turn, an increase in the lifetime of a particularcell can increase protein production from a cell culture. Apoptoticevents can be inhibited in a cell by introducing into a cell (such as amammalian cell, an insect cell, or a yeast cell) one or moreanti-apoptotic proteins, which inhibit apoptosis in cells at precisepoints along the apoptotic pathway. Another method to inhibit apoptosisis to inhibit release of pro-apoptotic molecules from the mitochondriain the cell. Variants of pro-apoptotic proteins known in the art, suchas a dominant-negative form of caspase-9, can be used as an inhibitor ofapoptosis in a cell. Such a variant protein can be introduced into acell in order to delay programmed cell death. Inhibition of apoptosis ofa cell, in turn, prolongs the time during which a particular cellproduces protein, resulting in an overall increase in the production ofa desired protein by a particular cell. Several genes that encodecaspase inhibitors (such as X-linked inhibitor of apoptosis (XIAP) orvariants therof) or anti-apoptotic genes (for example, Bcl-2 andBcl-x_(L) or variants thereof), can be transfected into geneticallyengineered mammalian cells (such as, CHO cells, VERO cells, BHK cells,and the like) (Sauerwald, T. et al., 2003, Biotechnol Bioeng.81:329-340; Sauerwald, T. et al., 2002, Biotechnol Bioeng. 77:704-716;Mastrangelo, A., et al., 2000, Biotechnol Bioeng. 67:544-564; Kim, N.,et al., 2002, J Biotechnol. 95:237-248; Figueroa, B., et al., 2001,Biotechnol Bioeng. 73:211-222).

In one embodiment, mammalian cells, which produce a recombinant protein,can be transfected with a vector containing an anti-apoptotic gene (suchas bcl-2). In another embodiment, recombinant mammalian cells can betransfected with a plasmid that contains a gene encoding for a caspaseinhibitor, or a gene that encodes a variant of a pro-apoptotic moleculeas described above, a gene that encodes a protein that is known to anindividual skilled in the art to possess anti-apoptotic activity, or anycombination thereof

In another embodiment, the overall product quality (for example enhancedglycosylation) of a desired recombinant protein (such as a therapeuticprotein) can be enhanced. To increase glycosylation of a recombinantprotein, mammalian cells (for example CHO cells, VERO cells, BHK cells,and the like) can be transfected with nucleic acids encoding one or moreenzymes that are involved in glycosylation (such asα2,3-sialyltransferase, β1,4-galactosyltransferase, and the like) ofproteins (Weikert et al., 1999, Nature Biotechnol 17:1116-21). In oneembodiment, a plasmid that encodes β1,4-galactosyltransferase can beintroduced into mammalian cells expressing a protein of interest. Inanother embodiment, a plasmid that encodes α2,3-sialyltransferase can beintroduced into mammalian cells expressing a protein of interest.

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland et al., J. Immunol. Methods, 56:221-234 (1983)) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)), andvary according to the particular host cell selected.

Without limitation, cell culture medium (such as inoculum medium, feedmedium, basal medium, and the like) can refer to a nutrient solutionused for growing and or maintaining cells, especially mammalian cells.These solutions ordinarily provide at least one component from one ormore of the following categories: (1) an energy source, usually in theform of a carbohydrate such as glucose; (2) all essential amino acids,and usually the basic set of twenty amino acids plus cysteine; (3)vitamins and/or other organic compounds required at low concentrations;(4) free fatty acids or lipids, for example linoleic acid; and (5) traceelements, where trace elements are defined as inorganic compounds ornaturally occurring elements that are typically required at very lowconcentrations, usually in the micromolar range. The nutrient solutioncan be supplemented electively with one or more components from any ofthe following categories: (1) hormones and other growth factors such as,serum, insulin, transferrin, and epidermal growth factor; (2) salts, forexample, magnesium, calcium, and phosphate; (3) buffers, such as HEPES;(4) nucleosides and bases such as, adenosine, thymidine, andhypoxanthine; (5) protein and tissue hydrolysates, for example peptoneor peptone mixtures which can be obtained from purified gelatin, plantmaterial, or animal byproducts; (6) antibiotics, such as gentamycin; (7)cell protective agents, for example pluronic polyol; and (8) galactose.An example of basal medium can be Cell Growth Basal Medium. An exampleof incoculum medium can be Inoculum Cell Growth Basal Medium. An exampleof feed medium can be Production Bioreactor Feed Medium.

Commercially available media can be utilized and include, for example,Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco'sModified Eagles Medium (DMEM, Sigma); Ham's F10 Medium (Sigma); HyClonecell culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma);and chemically-defined (CD) media, which are formulated for particularcell types, e.g., CD-CHO Medium (Invitrogen, Carlsbad, Calif.). Any ofthese media can be supplemented as necessary with the previously definedsupplementary components or ingredients, including optional components,in appropriate concentrations or amounts, as necessary or desired. Themammalian cell culture that can be used with the present invention isprepared in a medium suitable for the particular cell being cultured. Inone embodiment, the cell culture medium can be one of the aforementionedthat is generally free of serum from any mammalian source (for example,fetal bovine serum (FBS)). In another embodiment of this invention, themammalian cell culture can be grown in the commercially availablechemically defined (CD)-CHO Medium, supplemented with additionalcomponents specified in Table 15. In a further embodiment, the mammaliancell culture can be grown in CD-CHO Medium, supplemented with additionalcomponents specified in Table 20 or 21.

The methods of the present invention include the culturing of numerouscell types. In one embodiment of the invention, the cells are animal ormammalian. In another embodiment, the cells can express and secretelarge quantities of a desired protein. In another embodiment of theinvention, cells can express and secrete large quantities of aglycoprotein of interest into the culture medium. The animal ormammalian cells can also be molecularly modified to express and secretea protein of interest. The protein produced by the host cell can beendogenous or homologous to the host cell. The protein also can beheterologous (for example, foreign), to the host cell whereby geneticinformation coding for the protein of interest is introduced into thehost cell via methods standard in the art (for example byelectroporation, transfection, and the like). In one embodiment, amammalian glycoprotein can be produced and secreted by a Chinese hamsterovary (CHO) host cell into the culture medium.

In some embodiments, the invention provides populations of CTLA4-Igmolecules produced by the methods of production discussed herein,including the method of mass-production that is described in Example 14.The process can result in the production of CTLA4-Ig molecules of highmolecular weight (HMW) (for example, see Examples 14 and 15). In anotherembodiment, populations of CTLA4^(A29YL104E)-Ig molecules are providedthat are produced by the production methods discussed herein, such asthe method of mass-production that is described in EXAMPLES 19 and 20,and shown in FIG. 23. The process can result in the production ofCTLA4^(A29YL104E)-Ig molecules of high molecular weight (HMW) (forexample, see EXAMPLES 19 and 20). In some embodiments, the HMW speciescan be about 15-25% of the molecules or dimer produced by a method forproduction, including a chemically defined (CD)-CHO1 fermentationprocess. In other embodiments, the present invention provides methodsfor isolation, purification and characterization of CTLA4-Ig orCTLA4^(A29YL104E)-Ig HMW components produced by a CD-CHO1 fermentationprocess. CTLA4-Ig or CTLA4^(A29YL104E)-Ig HMW components are multimers(i.e, tetramers, hexamers, etc.), which have a higher molecular weightthan CTLA4-Ig or CTLA4^(A29YL104E)-Ig dimers.

Animal or mammalian host cells capable of harboring, expressing, andsecreting large quantities of a glycoprotein of interest into theculture medium for subsequent isolation and/or purification include, butare not limited to, Chinese hamster ovary cells (CHO), such as CHO-K1(ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet,12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/dhfr-,Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), anddp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cellstransformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonickidney cells (e.g., 293 cells, or 293 cells subcloned for growth insuspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); babyhamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCCCCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587;VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather, 1980, Biol.Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2);canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCCCCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumorcells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCCCRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68);MCR 5 cells; FS4 cells. In one aspect of this invention, CHO cells areutilized, particularly, CHO/dhfr-and CHO DG44 cells.

Examples of mammalian glycoproteins that can be produced by the methodsof this invention include, without limitation, cytokines, cytokinereceptors, growth factors (e.g., EGF, HER-2, FGF-α, FGF-β, TGF-α, TGF-β,PDGF, IGF-1, IGF-β); growth factor receptors, including fusion orchimeric proteins. Other examples include, but are not limited to growthhormones (e.g., human growth hormone, bovine growth hormone); insulin(e.g., insulin A chain and insulin B chain), proinsulin; erythropoietin(EPO); colony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF);interleukins (e.g., IL-1 through IL-12); vascular endothelial growthfactor (VEGF) and its receptor (VEGF-R); interferons (e.g., IFN-α, β, orγ); tumor necrosis factor (e.g., TNF-α and TNF-β) and their receptors,TNFR-1 and TNFR-2; thrombopoietin (TPO); thrombin; brain natriureticpeptide (BNP); clotting factors (e.g., Factor VIII, Factor IX, vonWillebrands factor, and the like); anti-dotting factors; tissueplasminogen activator (TPA), e.g., urokinase or human urine or tissuetype TPA; follicle stimulating hormone (FSH); luteinizing hormone (LH);calcitonin; CD proteins (e.g., CD3, CD4, CD8, CD28, CD19, etc.); CTLAproteins (e.g., CTLA4); T-cell and B-cell receptor proteins; bonemorphogenic proteins (BNPs, e.g., BMP-1, BMP-2, BMP-3, etc.);neurotrophic factors, e.g., bone derived neurotrophic factor (BDNF);neurotrophins, e.g., 3-6; renin; rheumatoid factor; RANTES; albumin;relaxin; macrophage inhibitory protein (e.g., MIP-1, MIP-2); viralproteins or antigens; surface membrane proteins; ion channel proteins;enzymes; regulatory proteins; antibodies; immunomodulatory proteins,(e.g., HLA, MHC, the B7 family); homing receptors; transport proteins;superoxide dismutase (SOD); G-protein coupled receptor proteins (GPCRs);neuromodulatory proteins; Alzheimer's Disease associated proteins andpeptides, (e.g., A-beta), as well as others known in the art. Suitableproteins, polypeptides, and peptides that can be produced by the methodsof the present invention include, but are not limited to, fusionproteins, polypeptides, chimeric proteins, as well as fragments orportions, or mutants, variants, or analogs of any of the aforementionedproteins and polypeptides.

The methods of the invention can also be used to produce CTLA4-Igmolecules which are variants of SEQ ID NO: 5, 6, 7, 8, 9, or 10. In oneembodiment, a CTLA4-Ig molecule can comprise a monomer having one ormore changes in residues 55 (ASSY) and 130 (L130E) (residues referred toare from SEQ ID NO:2). See the descriptions of variants and mutants ofCTLA4-Ig described in U.S. Publication No. US 2002/0182211 Al, which ishereby incorporated by reference in its entirety. In another embodiment,a CTLA4-Ig variant can comprise a CTLA4-Ig molecule having a mutationwithin the CTLA-4 region or a mutation in the Ig region, or anycombination thereof. In one embodiment, a CTLA4-Ig variant moleculecomprises a CTLA4-Ig molecule having an amino acid sequence that is atleast about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 99% identical to SEQ ID NOS: 5, 6, 7, 8, 9, or 10. In oneembodiment, the CTLA4-Ig variant molecule is capable of binding to CD80or CD86. In another embodiment, the variant is able to form a dimer. Ina further embodiment, the variant exhibits a carbohydrate profilesimilar to that exhibited by a non-mutated CTLA4-Ig molecule population.In one embodiment, the CTLA4-Ig variant molecules have the samepotential N-linked and O-linked glycosylation sites present in SEQ IDNO:2. In another embodiment, a CTLA4-Ig variant molecule has the sameN-linked and O-linked glycosylation sites present in SEQ ID NO:2, andhas additional glycosylation sites. The mutations can include, but arenot limited to, nucleotide deletions, insertions, additions; amino aciddeletions, substations, additions; nucleic acid frameshifts; thesubstitutions can be either non-conservative (e.g., a glycinesubstituted with a tryptophan) or conservative substitutions (e.g., aleucine substituted for an isoleucine).

CTLA4-Ig variant molecules include, but are not limited to,CTLA4-L104EA29YIg (using the residue numbering system according to SEQID NO:2, CTLA4-L104EA29YIg herein is referred to as CTLA4-L130EA55YIg),as well as those CTLA4-Ig variant molecules described in U.S. patentapplication Ser. Nos. 09/865,321 (U.S. Pub. No. US2002/0182211),60/214,065 and 60/287,576; in WO 01/92337 A2; in U.S. Pat. Nos.6,090,914, 5,844,095 and 5,773,253; and as described in R. J. Peach etal., 1994, J Exp Med, 180:2049-2058. In one embodiment, CTLA4-Ig variantmolecules produced in the present methods can be secreted from a cellthat comprises an expression vector coding for a CTLA4-Ig variantprotein.

A CTLA4-Ig variant, L130EA55Yig, is a genetically engineered fusionprotein similar in structure to CTAL4-Ig molecule. L130EA55Y-Ig has thefunctional extracellular binding domain of modified human CTLA-4 and theFc domain of human immunoglobulin of the IgGl class. Two amino acidmodifications, leucine to glutamic acid at position 104 (L104E) of anL104EA29Y variant, which corresponds to position 130 of SEQ ID NO:2, andalanine to tyrosine at position 29 (A29Y) of an L104EA29Y variant, whichcorresponds to position 55 of SEQ ID NO:2, were made in the B7 bindingregion of the CTLA-4 domain to generate L130EA55Y. L130EA55Y-Ig cancomprise two homologous glycosylated polypeptide chains of approximately45,700 Daltons each, which are held together by one inter-chaindisulfide bond and non-covalent interactions. DNA encoding L130EA55Y-Igwas deposited as DNA encoding L104EA29Y-Ig on Jun. 20, 2000, with theAmerican Type Culture Collection (ATCC) under the provisions of theBudapest Treaty. It has been accorded ATCC accession number PTA-2104.L104EA29Y-Ig (corresponding to L130EA55Y-Ig in this application) isfurther described in co-pending U.S. patent application Ser. Nos.09/579,927, 60/287,576 and 60/214,065, and 09/865,321 and inWO/01/923337 A2, all of which are incorporated by reference in thisapplication in their entireties.

Since the recombinant protein L130EA55Y-Ig is different at only 2 aminoacids (Tyr at amino acid position 55 and Glu at amino acid position 130)compared to CTLA4-Ig monomers having an Ala at amino acid position 55and Leu at amino acid position 130 of SEQ ID NO:2, and because these 2mutations do not affect N-or O-linked glycosylation, CTLA4-Ig variantmolecule populations comprising L130EA55Y-Ig may have the same profileor a very similar glycosylation profile as do populations comprisingwild type CTLA4-Ig. Further, because the recombinant proteinL130EA55Y-Ig is different at only 2 amino acids (Tyr at amino acidposition 55 and Glu at amino acid position 130) compared to CTLA4-Igmonomers having an Ala at amino acid position 55 and Leu at amino acidposition 130 of SEQ ID NO:2, the present methods of this inventionshould be able to produce L130EA55Y-Ig with similar characteristicattributes as described in Table 6.

The methods of the invention can also be used to produceCTLA4^(A29YL104E)-Ig molecules, which are variants of SEQ ID NOS: 11,12, 13, 14, 15, or 16. In one embodiment, a CTLA4^(A29YL104E)-Ig cancomprise a monomer having one or more changes in SEQ ID NO:3. Forexample, descriptions of other CTLA4^(A29YL104E)-Ig molecules aredescribed in U.S. Patent Application Publication Nos. U.S. 2002/0039577,U.S. 2003/0007968, U.S. 2004/0022787, U.S. 2005/0019859, and U.S.2005/0084933, and U.S. Pat. No. 7,094,8874, which are herebyincorporated by reference in their entirety.

In one embodiment, CTLA4^(A29YL104E)-Ig comprises one or more mutationswithin the CTLA-4 region (SEQ ID NO:18), or a mutation in the Ig region,or any combination thereof. In other embodiments, a CTLA4^(A29YL104E)-Igmolecule comprises a CTLA4^(A29YL104E)-Ig having an amino acid sequencethat is at least about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 99% identical to SEQ ID NOS: 11, 12, 13, 14, 15, or16. In a further embodiment, a CTLA4^(A29YL104E)-Ig molecule asdescribed above is capable of binding to CD80 or CD86. In anotherembodiment, the CTLA4^(A29YL104E)-Ig is able to form a dimer. In afurther embodiment, the CTLA4^(A29YL104E)-Ig exhibits a carbohydrateprofile similar to that exhibited by a non-mutated CTLA4^(A29YL104E)-Igmolecule population. In yet other embodiments, the CTLA4^(A29YL104E)-Igmolecules have the same potential N-linked and O-linked glycosylationsites present in SEQ ID NO:4. In another embodiment, aCTLA4^(A29YL104E)-Ig has the same N-linked and O-linked glycosylationsites present in SEQ ID NO:4, and has additional glycosylation sites.The mutations can include, but are not limited to, nucleotide deletions,insertions, additions; amino acid deletions, substitutions, additions;nucleic acid frameshifts; the substitutions can be eithernon-conservative (e.g., a glycine substituted with a tryptophan) orconservative substitutions (e.g., a leucine substituted for anisoleucine).

In one embodiment of the invention, a population of CTLA4-Ig variantmolecules can be produced by mammalian cells (for example dhfr negativeCHO cells), which express a gene encoding the desired protein (forexample a L130EA55Y-Ig protein or the like), grown in suspensionaccording to the mass-production method of this invention. According tothis invention, a recombinant CTLA4-Ig variant protein produced bymammalian cells can be recovered according to the harvesting parametersdescribed herein. In other embodiments, a recombinant CTLA4-Ig variantprotein produced by mammalian cells can be purified according to thepurification scheme described in this invention (Example 15).

In one embodiment of the invention, a population of CTLA4^(A29YL104E)-Igmolecules can be produced by mammalian cells (for example dhfr negativeCHO cells), which express a gene encoding the desired protein (forexample a CTLA4^(A29YL104E)-Ig), grown in suspension according to themass-production method of this invention. According to this invention, arecombinant CTLA4^(A29YL104E)-Ig produced by mammalian cells can berecovered according to the harvesting parameters described herein. Inother embodiments, a recombinant CTLA4^(A29YL104E)-Ig produced bymammalian cells can be purified according to the purification schemedescribed in this invention (EXAMPLES 19-20).

Types of Cell Cultures and General Culturing Processes

A protein of interest, for example a glycoprotein, a fusion protein andthe like, can be produced by growing cells expressing the desiredprotein product under a variety of cell culture conditions. Apractitioner skilled in the art understands that cell cultures andculturing runs for protein production can include, but are not limitedto, three general types: continuous culture, batch culture, andfed-batch culture. In a continuous culture process, a fresh culturemedium supplement (for example, feeding medium) is supplied to cellsduring the culturing period while old culture medium is removed. Theproduct produced during a continuous culture can also be harvested, forexample, on a daily basis or continuously. As long as the cells remainalive, and the environmental and culturing conditions are maintained,cells can remain in culture as long as is desired in a continuousculturing process.

In a batch culture process, cells are initially cultured in medium andthis culturing medium is neither replaced, nor removed, norsupplemented. The cells are not “fed” with new medium during or beforethe end of the culturing run thus culturing continues until nutrientsare exhausted. The protein product is harvested at the end of theculturing run.

For fed-batch culture processes, the culturing run time can be increasedby supplementing the culture medium one or more times daily (orcontinuously) with fresh medium during the run. In this process, thecells are supplied with fresh medium, a “feeding medium”, during theculturing period. Fed-batch cultures can include the various feedingschedules described previously, for example, daily, every two days,every other day, etc.; more than once per day, or less than once perday, and so on. Fed-batch cultures also can be fed continuously withfeeding medium. At the end of the culturing/production run, the proteinproduct of interest is then harvested.

Cell culture systems for the small-or large-scale production ofproteins, including glycoproteins, produced by mammalian host cells areuseful within the context of this invention. Those having skill in theart understand that tissue culture dishes, spinner flasks, and T-flasksare typically used for culturing methods on a laboratory scale. Theprocesses that can be used for culturing on a larger scale (e.g., 500 L,5000 L, 10,000 L, 20,000 L and the like) include, but are not limitedto, a hollow fiber bioreactor, a fluidized bed bioreactor, a stirredtank bioreactor system, or a roller bottle culture. The later twoprocesses can be utilized with or without microcarriers.

The systems can be operated in a batch, fed-batch, or continuous mode.For production-scale culturing, the stirred-tank bioreactor is thesystem of choice because of its flexibility. These reactors can maintaincells in suspension by agitation through mechanical stirring with gasbubble sparging or an impeller. The stirred-tank bioreactors can bescaled up to large production-scale volumes (for example, 20,000 liters)and can be operated in different feed modes. These systems provide alarge surface area for cell growth and the efficient transfer ofmetabolic wastes, oxygen, and nutrients, as well as maintain ahomogenous environment throughout the reactor by preventing cells fromsettling to the bottom via continuous stirring or mixing of thecomponents within the reactors. For the production of a desiredglycoprotein, the present invention embodies large-scale, fed-batch cellcultures maintained in a stirred-tank bioreactor, fed daily with feedingmedium containing D-galactose. In another embodiment, fed-batch cellcultures can also be maintained in a stirred-tank bioreactor, fed dailywith feeding medium that contains suitable concentrations of thelimiting cell culture nutrients important for protein glycosylation,such as glucose and glutamine (Chee et al., 2005, Biotechnol. Bioeng.89:164-177).

The cells of the culture producing a protein of interest can bepropagated according to any scheme or routine that is most suitable forthe particular mammalian host cell and the particular production plancontemplated. Cell culture conditions can be developed to enhanceexpansion or growth of a population of mammalian host cells in thegrowth phase of the cell culture for a period of time that is maximizedfor such expansion and growth. The growth phase of the cell culturecomprises the period of exponential cell growth (for example, the logphase) where cells are primarily dividing rapidly. During this phase,the rate of increase in the density of viable cells is higher than atany other time point.

Also, cell culture conditions can be developed to enhance proteinproduction during the production phase of the cell culture for a periodof time. The production phase of the cell culture comprises the periodof time during which cell growth is stationary or is maintained at anear constant level. The density of viable cells remains approximatelyconstant over a given period of time. Logarithmic cell growth hasterminated and protein production is the primary activity during theproduction phase. The medium at this time is generally supplemented tosupport continued protein production and to achieve the desiredglycoprotein product.

Culture conditions, such as temperature, pH, dissolved oxygen (DO₂), andthe like, are those used in culturing mammalian host cells that areunderstood by the individual skilled in the art. An appropriatetemperature range for culturing mammalian host cells, such as CHO cells,is between 30 to 40° C., and in one embodiment about 37° C. The pHgenerally is adjusted to a level between about 6.5 and 7.5 using eitheran acid or base. A suitable DO₂ is between 5-90% of air saturation.These culture conditions can be used to facilitate the culturing ofmammalian cells that produce a desired protein or glycoprotein product.

A mammalian host cell population can be expanded and grown in a growthphase culture wherein cells, possibly removed from storage, areinoculated into a culturing medium acceptable for promoting growth andhigh viability. The cells can then be maintained in a production phasefor a suitable period of time by the addition of fresh culturing mediumto the host cell culture. During the production phase, cells can besubjected to various shifts in temperature to enhance proteinproduction. Multiple temperature shift culturing processes are describedin patent applications U.S. Ser. No. 10/742,564, filed Dec. 18, 2003,and U.S. Ser. No. 10/740,645, filed on Dec. 18, 2003. The contents fromthese applications are incorporated by reference herein in theirentirety. In this invention, the two or more temperature shiftscomprising the cell culture processes can result in an increased numberof viable cells that survive in culture until the end of the process orproduction run. During the production phase of the culture, the greaterthe number of cells that survive can result in a greater amount ofprotein or glycoprotein product produced, increasing the amount ofprotein product at the end of the process.

A particular aspect of this invention embodies a fed-batch, large-scale(for example 500 L, 5000 L, 10000 L, and the like), mammalian cellculture, that is fed daily or with feeding medium described in Tables14, 15, comprising D-galactose in order for cells to produce aglycoprotein of interest. To increase the quality of the proteinproduced in this embodiment, two or more temperature shifts can beemployed during the culture period to extend the protein productionphase beyond that which occurs when no temperature shift is used, orwhen only one temperature shift is used. In another embodiment, theinvention entails a fed-batch, large-scale (for example 500 L, 5000 L,10000 L, and the like), mammalian cell culture, that is fed 1 or moretimes daily with feeding medium described in Table 22, which compriseD-galactose in order for cells to produce a glycoprotein of interest(for example, CTLA4^(A29YL104E-)Ig). One or more temperature shifts alsocan be employed during the culture period to extend the proteinproduction phase beyond that which occurs when no temperature shift isused in order to increase the quality of the glycoprotein.Alternatively, dextran sulfate can be added to the culture with aconcomitant temperature shift.

Mass-Production of Recombinant Protein in Bioreactors

The present invention provides methods for conventional stirred tankbioreactor cultivation of eukaryotic cells (for example, a 20000 L cellculture volume), particularly to produce large-scale or industrialamounts of desired protein products that are expressed by such cells.The cultivation process is a fed-batch culturing process of eukaryoticcells grown in suspension, with harvesting of culture supernatant,wherein eukaryotic cells, for example mammalian cells, expressing aprotein of interest, secrete desired protein product into the culturemedium.

Methods for large-scale cultivation of mammalian cells, particularly toproduce large amounts of desired protein products that are expressed bysuch cells, are embodied in the present invention. The methods can becarried out by steps comprising:

(i) inoculating cells into a seed culture vessel (for example, a T-175flask) containing serum-free culture medium and propagating the seedculture (for example, a starter culture that used to inoculate a largervolume) at least until the cells reach a minimal cross-seeding densitywhereby the density is a pre-determined value needed for sufficientpropagation of cells in the subsequent culturing volume;

(ii) transferring the propagated seed culture to a larger culture vessel(for example, roller bottles or cell bags) containing culture mediumlacking animal-derived components in order to expand the culture;

(iii) transferring the expanded seed culture to a large-scale culturevessel containing serum-free culture medium to further propagate to thecell culture; and

(iv) maintaining the large-scale culture in medium lackinganimal-derived components, at least until said cells reach a targetdensity or display a specific biochemical characteristic.

In some embodiments, the method can comprise the step of (iv) harvestingof the culture medium and replacing that medium with fresh medium.

In other embodiments, methods for large-scale cultivation of mammaliancells can be carried out by steps comprising:

(i) inoculating cells into a seed culture vessel (for example, a T-175flask) containing serum-free culture medium (for example, inoculoummedium) and propagating the seed culture (for example, a starter culturethat used to inoculate a larger volume) at least until the cells reach aminimal cross-seeding density whereby the density is a pre-determinedvalue needed for sufficient propagation of cells in the subsequentculturing volume;

(ii) transferring the propagated seed culture to a larger culture vessel(for example, roller bottles or cell bags) containing culture mediumlacking animal-derived components (for example, inoculum medium) inorder to expand the culture;

(iii) transferring the expanded seed culture to a large-scale culturevessel (such as 1000-L bioreactors) containing serum-free culture medium(for example, basal medium) to further propagate to the cell culture;and

(iv) maintaining the large-scale culture in medium lackinganimal-derived components (for example, feed medium), at least untilsaid cells reach a target density or display a specific biochemicalcharacteristic.

In some embodiments of the invention, the method can comprise the stepof:

(v) harvesting of the culture medium and replacing the spent medium withfresh medium.

The present invention is applicable to any cell type in any formulationof medium lacking animal-derived components in order to producelarge-scale quantities of desired protein products, and can utilizeeither of the following two processes, or variations thereof:

a) microcarrier processes, or b) suspension cell processes. Culturing ofcells, for example mammalian cells, can utilize either process, operatedin two distinct phases, a growth phase and a production phase. Inanother embodiment of the invention, any formulation of medium whichcontains animal-derived components (some non-limiting examples beingBovine-Serum Albumin (BSA) or FBS) can be employed as well for theproduction of large-scale protein quantities as described above.

One skilled in the art understands that a microcarrier process, notlimited to a standard microcarrier-process or a perfusion microcarrierprocess, can be used for cell culturing wherein cells are attached toand/or immobilized in a macroporous carrier. In a standardmicrocarrier-process, cells are inoculated into a seed culture vesselcontaining serum-free culture medium and propagated until the cellsreach a minimum seeding density. Subsequently, the propagated seedculture is transferred to a large-scale culture vessel containingserum-free culture medium and microcarriers. In this growth phase, thecells are grown on microcarriers until the carriers are fully colonized,for example by cells migrating into the carriers in the case of aprocess using macroporous carriers.

Medium exchange can occur when microcarriers settle to the bottom of theculture vessel, after which a predetermined percentage of the tankvolume is removed and a corresponding percentage tank volume of freshmedium is added to the vessel. Microcarriers are then re-suspended inthe culturing medium. A skilled artisan understands that the process ofmedium removal and replacement can be repeated at a predeterminedinterval, for example every 24 hours whereby the amount of replacedmedium is dependent on cell density and can typically be from 25% to 80%of the tank volume. 60-95% of the tank medium in the tank can be changedevery 24 hours when the cell density reaches a pre-determined valuesuitable for protein expression. Those having skill in the art often usethe aforementioned medium exchange % value throughout the productionphase as well.

During the production phase, culture medium can be exchanged by allowingthe microcarriers to settle to the bottom of the tank, after which theselected % of the tank volume is removed and a corresponding % tankvolume, for example 60-95% as described earlier, of fresh culturingmedium is added to the vessel. Microcarriers are then re-suspended inthe culturing medium and the medium removal and replacement process canbe repeated daily.

The microcarrier perfusion process resembles the standard microcarrierprocess and also is operated in the growth/expansion and productionphases. The main difference between the two processes is the methodemployed to change the culture medium. A defined amount of the tankvolume, for example 60-95% of the total tank volume, is changed all atonce in the standard microcarrier process, whereas in the perfusionprocess the medium is added continuously. Essentially, a % tank volumemedium is changed gradually over a predetermined length of time whilethe microcarriers are kept in the vessel by using a separation device(or perfusion device) that allows the medium to leave the vessel butretains the microcarriers within the tank. The growth phase in thisprocess is as described for a standard microcarrier process except forthe gradual medium exchange.

Two non-limiting options for a suspension cell process include asuspension cell perfusion process and a suspension cell batch process.In the perfusion process, cells in a culturing medium are freelysuspended without being immobilized in carriers and, and like themicrocarrier processes, can be operated in two distinct phases (forexample, a growth phase and a production phase). During the growth phaseof a suspension cell-perfusion process, cells are inoculated into a seedculture vessel containing serum-free culture medium and propagated untilcells reach a target cross-seeding density. The propagated seed culturecan then be transferred to a large-scale culture vessel, which containsculturing medium lacking animal-derived components, and propagated untila pre-determined cell density value suitable for protein expression isreached. A continuous perfusion of the culture vessel with fresh culturemedium is performed to execute the medium exchange process.

In the suspension cell batch process, cell culturing can be carried outvia the following non-limiting formats: a) simple batch process or b)fed-batch process. Cells are inoculated into a seed culture vesselcontaining culture medium lacking animal-derived components in a simplebatch process and propagated until the cells reach a pre-determinedcross-seeding density. Subsequently, the propagated seed culture istransferred to a large-scale culture vessel containing serum-freeculture medium and the culturing vessel is operated until the nutrientsin the culture medium have been exhausted. In a fed-batch process,feeding a concentrated solution of nutrients (for example a feed medium)to the tank can extend the nutrient supply in the medium of thisculturing process, thus extending the process time and ultimatelyleading to an increase in the production of the desired protein withinthe culture vessel. The method of adding the feed medium can vary. Itcan be added either as a single pulse bolus (once, twice, three timesetc., a day) or can be fed gradually throughout a 24-hour period. Thisfeed allows cells to be propagated in a large-scale culture vessel andthe medium, which can contains the secreted protein product of interest,to be harvested at the end of the run before any of the nutrients becomeexhausted. Instead of removing all of the contents from the vessel, oneskilled in the art would remove only a portion of the tank volume (canbe about 80%).

An optional aspect of the fed-batch process is the use of temperatureshifts. In this process, temperatures employed as the operatingtemperatures during the production phase are lower than the temperatureused during the growth phase. Said temperature ranges for a fed batchprocess, for example the process used in this invention, could consistof an initial growth phase at a temperature suitable for growth of theparticular cell line in use followed by a decrease in the operatingtemperature at a pre-determined cell density.

In one embodiment, a process for a large-scale fed-batch culture processcomprises the following: (i) inoculating cells into a seed culturevessel (for example, a T-175 flask) containing serum-free culture mediumand propagating the seed culture at least until the cells reach apre-determined cross-seeding density at a temperature suitable forgrowth; (ii) transferring the propagated seed culture to a largerculture vessel (for example, roller bottles or cell bags) containingculture medium lacking animal-derived components in order to expand theculture at a suitable temperature suitable; (iii) transferring theexpanded seed culture to a large-scale culture vessel containingserum-free culture medium to further propagate to the cell culture at asuitable temperature; and (iv) maintaining the large-scale culture at adecreased temperature suitable for protein expression, in medium lackinganimal-derived components, with daily replacements by fresh feed medium,at least until said cells reach a target density or critical length oftime.

The step of replacement with fresh feed medium in (iv) can entailremoving a predetermined volume, for example 80%, of the tank volume andreplacing it with the same volume of fresh feed medium.

In a further embodiment, a process for a large-scale fed-batch cultureprocess comprises the following: (i) inoculating cells into a seedculture vessel (for example, a T-175 flask) containing serum-freeculture medium and propagating the seed culture at least until the cellsreach a pre-determined cross-seeding density at a temperature suitablefor growth; (ii) transferring the propagated seed culture to a largerculture vessel (for example, roller bottles or cell bags) containingculture medium lacking animal-derived components in order to expand theculture at a suitable temperature suitable; (iii) transferring theexpanded seed culture to a large-scale culture vessel (for example, a1000-L bioreactor) containing serum-free culture medium to furtherpropagate to the cell culture at a suitable temperature; and (iv)maintaining the large-scale culture at a decreased temperature suitablefor protein expression, in medium lacking animal-derived components,with daily replacements by fresh feed medium, at least until said cellsreach a target density or critical length of time.

The step of replacement with fresh feed medium in (iv) can entailremoving a predetermined volume, for example about 80% of the tankvolume, and replacing it with the same volume of fresh feed medium.

In one embodiment of this invention, the cells cultured in a fed-batchprocess are mammalian cells, for example CHO cells, which express adesired protein product. Mammalian cells are inoculated into a seedculture vessel (for example, a T-175 flask) containing serum-freeculture medium, for example CD-CHO medium (Example 13), and propagatedat a temperature suitable for growth, for example at about 35-39° C.,for 3-4 days until the cells reach a pre-determined cross-seedingdensity (for example, having ≥6.0×10⁶ viable cells, or wherein the finalculture viability ≥80%). The propagated seed culture is then transferredto a large culture vessel (for example, roller bottles) containingculture medium lacking animal-derived components for expansion at asuitable temperature (for example at about 35-39° C.) for approximately3-4 days. The cell culture is further expanded in a larger culturevessel (for example, a 20 L cell bag, a 100 L cell bag, and the like)containing serum free medium, for example CD-CHO medium, at atemperature suitable for growth, for example at about 35-39° C., for 3-4days until the cells reach a target seeding density (for example, having≥1-2×10⁶ viable cells/ml, or wherein the final culture viability≥80%).In one embodiment, the inoculum expansion involves a minimum of 4passages. In another embodiment of the invention, inoculum expansionentails no more than 20 passages.

The expanded seed culture can then used to inoculate a large-scaleculturing tank (for example, a 1000 L, a 4000 L bioreactor and thelike), containing serum-free culture medium (for example CD-CHO medium)to further propagate the cell culture at a suitable temperature, forexample at about 35-39° C., for 3-6 days, until the cells reach a targetseeding density (for example, having ≥1-2×10⁶ viable cells/ml, orwherein the final cell culture viability ≥80%). A large-scale culture(for example a 10,000 L, 15,000 L, 20,000 L culture in a bioreactor andthe like) is subsequently maintained in serum-free culture medium,wherein the medium is a feed medium (for example eRDF medium, Example14), at a temperature lower than the growth temperature (for example ator about 33-35° C. for 3-4 days, and at or about 31-33° C. for 6-8days), suitable for protein expression and production of the secretedprotein product. The feed medium is replaced daily with fresh feedmedium, whereby the tank's replacement with fresh feed medium entailsremoval of a predetermined volume, for example 80% of the tank volume,and replacing the tank with the same volume of fresh feed medium. Thecommercial scale culture is maintained until said cells reach a targetvalue of production parameters that can be, but are not limited to, alength of time, a target cell density, or biochemical proteincharacteristic (such as a NANA molar ratio as previously described)wherein the viable cell density can be 3.0-8.0×10⁶ cells/ml; a NANAmolar ratio can be ≥6.0; a final cell culture viability can be ≥30%; anda final protein product titer can be ≥0.5 g/L).

In a particular embodiment of this invention, the cells cultured in afed-batch process are mammalian cells, for example CHO cells, whichexpress a desired protein product (for example, a CTLA4-Ig molecule).CHO cells are inoculated into a seed culture vessel (for example, aT-175 flask) containing serum-free culture medium, for example CD-CHOmedium, and propagated at a temperature suitable for growth, for exampleat about 3TC, for 3-4 days until the cells reach a pre-determinedcross-seeding density (for example, having ≥10.0×10⁶ viable cells, orwherein the final culture viability ≥84%). The propagated seed cultureis then transferred to a large culture vessel (for example, rollerbottles) containing culture medium lacking animal-derived components forexpansion at a suitable temperature (approximately 37° C.) for about 4days. The cell culture is further expanded in a larger culture vessel(for example, a 20 L cell bag, a 100 L cell bag, and the like)containing serum-free medium, for example CD-CHO medium, for 4 days at atemperature suitable for growth (for example at about 37° C.) until thecells reach a target seeding density (for example, having ≥1-2×10⁶viable cells/ml, or wherein the final culture viability ≥91%). Theinoculum expansion can involve a minimum of 7 passages.

The expanded seed culture is then used to inoculate a large-scaleculturing tank (for example, a 4000 L bioreactor and the like),containing serum-free culture medium (for example CD-CHO medium) tofurther propagate the cell culture at a suitable temperature, forexample at about 37° C., for 5-6 days, until the cells reach a targetseeding density (for example, having ≥1-2×10⁶ viable cells/ml, orwherein the final cell culture viability ≥86%). A commercial-scaleculture (for example a 20,000 L culture in a bioreactor) is subsequentlymaintained in serum-free culture medium, wherein the medium is a feedmedium (for example eRDF medium), at a temperature lower than the growthtemperature, which is suitable for protein expression and production ofthe secreted protein product (for example, CTLA4-Ig). Thecommercial-scale culture is first lowered from about 37° C. to about 34°C. for 4 days, and then subjected to a second temperature shift bylowering the temperature from about 34° C. to about 32° C. for 8 days.The feed medium is replaced daily with fresh feed medium, wherebyreplacing the feed medium in the bioreactor tank entails removal of apredetermined volume, for example 80% of the tank volume, and replacingit with the same volume of fresh feed medium. The commercial scale ismaintained until said CHO cells and/or secreted protein product reach atarget value of the following non-limiting production parameters: aviable cell density of 4.0-7.0×10⁶ cells/ml; a NANA molar ratio ≥8.0; afinal cell culture viability ≥38%; and a final protein product titer of≥0.6 g/L.

In another embodiment of this invention, the cells cultured in afed-batch process are mammalian cells, for example CHO cells, whichexpress a desired protein product. Mammalian cells are inoculated into aseed culture vessel (for example, a T-175 flask) containing serum-freeculture medium, for example CD-CHO medium (EXAMPLE 19), and propagatedat a temperature suitable for growth, for example from about 35° C. toabout 39° C., for about 3-4 days; or from about 36° C. to about 38° C.,for about up to about 4 days until the cells reach a pre-determinedcross-seeding density (for example, having a cell density of greaterthan or equal to1.5×10⁶, or wherein the final culture viability isgreater than or equal to about 80%). The propagated seed culture is thentransferred to a large culture vessel (for example, roller bottles)containing culture medium lacking animal-derived components forexpansion at a suitable temperature (for example, from about 35° C. toabout 39° C., or from about 36° C. to about 38° C.) for about 3-4 daysor up to about 4 days. The cell culture is further expanded in a largerculture vessel (for example, a 20 L cell bag, a 100 L cell bag, and thelike) containing serum free medium, for example CD-CHO medium, at atemperature suitable for growth, for example from about 35° C. to about39° C., or from about 36° C. to about 38° C., for about 3-4 days or upto about 4 days until the cells reach a target seeding density (forexample, having at least about 1.5×10⁶ viable cells/ml, or wherein thefinal culture viability is greater than or equal to 80%). In oneembodiment, the inoculum expansion involves a minimum of 4 passages. Inanother embodiment of the invention, inoculum expansion entails no morethan 20 passages. In some embodiments, the CD-CHO medium is a CD-CHOinoculum medium.

The expanded seed culture can then be used to inoculate a large-scaleculturing tank (for example, a 1000 L, a 4000 L bioreactor, and thelike), containing serum-free culture medium (for example CD-CHO medium,such as CD-CHO inoculum medium and/or CD-CHO basal medium) to furtherpropagate the cell culture at a suitable temperature, for example fromabout 35° C. to about 39° C., or from about 36° C. to about 38° C. forfrom about 3 to about 6 days, or for from about 4 to about 5 days, orfor about 4.7 days, or for less than or equal to about 113 hours, untilthe cells reach a target seeding density (for example, having about2.3×10⁶ viable cells/ml, or wherein the final cell culture viability isat least about 88%).

A commercial-scale culture (for example a 10,000 L, 15,000 L, 20,000 L,30,000 L culture in a bioreactor and the like) is subsequentlymaintained in serum-free culture medium, wherein the medium is a feedmedium (for example, eRDF medium, EXAMPLE 19), at a temperature of fromabout 35° C. to about 39° C. for from about 3 to about 6 days, or fromabout 4 to about 5 days, suitable for protein expression and productionof the secreted protein product. Alternatively, a polyanionic compound(for example, such as dextran sulfate) can be added to a culturemaintained in serum-free culture medium, wherein the medium is a feedmedium (for example, eRDF medium, EXAMPLE 19) as described below and inU.S. Pat. App. Publication No. 2005/0019859, which is herebyincorporated by reference in its entirety. The culture can beconcomitantly subjected to a single step temperature lowering (forexample, at or about 32° C. to at or about 36° C. for from about 3 toabout 14 days, or for from about 10 to about 13 days, or for from about234 to about 304 hours.

In one embodiment of the invention, the culture can also beconcomitantly subjected to a multi-step temperature lowering (forexample, at or about 33° C. to at or about 35° C. for about 3-6 days,and at or about 3FC to at or about 33° C. for about 6-8 days). The abovedescribed processed are suitable for protein expression and productionof the secreted protein product.

The feed medium in the instances described above can be replaced daily(1, 2, 3, etc. times daily) or every few days with fresh feed medium.The tank's replacement with fresh feed medium entails removal of apredetermined volume, for example 80% of the tank volume, and replacingthe tank with the same volume of fresh feed medium. The commercial scaleculture is maintained until said cells reach a target value ofproduction parameters that can be, but are not limited to, a length oftime, a target cell density, or biochemical protein characteristic (suchas a NANA molar ratio as previously described) wherein the viable celldensity can be 3.0-8.0×10⁶ cells/ml; a NANA molar ratio can be ≥5.0, orabout 6, or from about 5.2 to about 7.6; a final cell culture viabilitycan be greater than or equal to about 30% or greater than or equal toabout 37%; and a final protein product titer can be from about 0.46 toabout 0.71 g/L, greater than or equal to 0.5 g/L, or greater than orequal to 20 g/L.

In accordance with the present invention, a cell culture processinvolving the delayed addition of polyanionic compound is provided. Theprocess comprises adding polyanionic compound to a cell culture at atime after inoculation (for example, during the growth phase or duringthe production phase of the culturing process). The delayed addition ofpolyanionic compound achieves increased cell viability. In oneembodiment, the invention is directed to a cell culturing process thatcomprises culturing host cells, which express a protein of interest, andadding polyanionic compound to the cell culture at a time afterinoculation.

Polyanionic compounds include, but are not limited to, dextran sulfate(available from Sigma-Aldrich, St. Louis, Mo.), heparin (available fromSigma-Aldrich), heparan sulfate (available from Sigma-Aldrich), mannansulfate, chondroitin sulfate (available from Sigma-Aldrich), dermatansulfate (available from Sigma-Aldrich), keratan sulfate (available fromSigma-Aldrich), hyaluronate (available from Sigma-Aldrich), poly(vinylsulfate) (available from Sigma-Aldrich), kappa-carrageenan (availablefrom Sigma-Aldrich), and suramin (available from Sigma-Aldrich). Thecompounds are readily available from the listed sources, or readilyobtainable through means known to one of skill in the art. Thesecompounds are frequently available in the form of a salt, including butnot limited to sodium salt, but may also be used in non-salt forms. Apolyanionic compound includes all forms thereof, including but notlimited to salt forms, such as sodium salts.

Particularly useful, non-limiting examples of polyanionic compounds ofthe invention include poysulfated compounds: dextran sulfate, heparin,heparan sulfate, mannan sulfate, chondroitin sulfate, dermatan sulfate,keratan sulfate, poly(vinyl sulfate), kappa-carrageenan, and suramin. Inone embodiment, the polyanionic compound is dextran sulfate. Dextransulfate may have an average molecular weight of 5,000 to 500,000 Da. Inanother embodiment of the invention, dextran sulfate having a molecularweight of 5,000 Da is used.

According to methods of the invention, polyanionic compound may be addedto the cell culture one time, two times, three times, or any number oftimes during the specified cell culture period (for example, at a timeafter inoculation, such as during the growth phase or the productionphase). One or more polyanionic compounds may be used in conjunction.For example, any given single addition of a polyanionic compound mayinclude the addition of one or more other polyanionic compounds.Similarly, if there is more than one addition of a polyanionic compound,different polyanionic compounds may be added at the different additions.Additional compounds and substances, including polyanionic compounds,may be added to the culture before, with, or after the addition ofpolyanionic compound, either during or not during the specified timeperiod. In a particular embodiment, there is a single, for example onetime, addition of polyanionic compound. In another embodiment, onepolyanionic compound is added.

Polyanionic compound may be added to the cell culture by any means.Means of adding polyanionic compound include, but are not limited to,dissolved in water, dissolved in culture medium, dissolved in feedmedium, dissolved in a suitable medium, and in the form in which it isobtained. In particular, polyanionic compound is added dissolved inwater. In accordance with the invention, polyanionic compound is addedto bring the concentration in the culture to an appropriate level. Asnon-limiting examples, polyanionic compound is added to a concentrationof 1-1000 mg/L, 1-200 mg/L, 1-100 mg/L, or 25-75 mg/L. Particularlyuseful concentrations of polyanionic compound added to the cell cultureinclude, but are not limited to, about 25-200 mg/L; about 25-100 mg/L;and about 50-100 mg/L. In one embodiment of the invention, theconcentration of polyanionic compound added to the culture is about 50mg/L. In another embodiment, the concentration of polyanionic compoundadded to the culture is about 100 mg/L.

Methods of the invention provide that the culture may be run for anylength of time after addition of polyanionic compound. The culture runtime may be determined by one of skill in the art, based on relevantfactors such as the quantity and quality of recoverable protein, and thelevel of contaminating cellular species (e.g. proteins and DNA) in thesupernatant resulting from cell lysis, which will complicate recovery ofthe protein of interest. In some embodiments of the cell culturingprocess, polyanionic compound is added at a time after inoculation (forexample, during the growth phase of the cell culture process or duringthe production phase of the cell culture process). Polyanionic compoundis added at a time after inoculation that is during on or about the endof the growth phase. In particular, polyanionic compound is added at atime after inoculation that is during the production phase, for example,at the onset of the production phase.

In a particular embodiment of this invention, the cells cultured in afed-batch process are mammalian cells, for example CHO cells, whichexpress a desired protein product (for example, a CTLA4^(A29YL104E)-Igmolecule). CHO cells are inoculated into a seed culture vessel (forexample, a T-175 flask) containing serum-free culture medium, forexample CD-CHO medium (such as CD-CHO inoculum medium), and propagatedat a temperature suitable for growth, for example at about 37° C., forabout 3-4 days until the cells reach a pre-determined cross-seedingdensity (for example, having ≥1.5×10⁶ viable cells, or wherein the finalculture viability ≥80%). The propagated seed culture is then transferredto a large culture vessel (for example, roller bottles) containingculture medium lacking animal-derived components for expansion at asuitable temperature (at about 37° C.) for about 4 days. The cellculture is further expanded in a larger culture vessel (for example, a20 L cell bag, a 100 L cell bag, and the like) containing serum-freemedium, for example CD-CHO medium (such as CD-CHO inoculum medium), forabout 4 days at a temperature suitable for growth (for example, at about37° C.) until the cells reach a target seeding density (for example,having ≥1.5×10⁶ viable cells, or wherein the final culture viability≥80%). The inoculum expansion can involve a minimum of 7 passages.

The expanded seed culture is then used to inoculate a large-scaleculturing tank (for example, a 1000-L, a 4000-L bioreactor, and thelike), containing serum-free culture medium (for example CD-CHO medium,such as CD-CHO basal medium) to further propagate the cell culture at asuitable temperature, for example at about 37° C., for about 5-6 days,until the cells reach a target seeding density (for example, havingabout 2.3×10⁶ viable cells/ml, or wherein the final cell cultureviability ≥88%). A commercial-scale culture (for example a 10,000 L,15,0000 L, or 20,000 L culture and the like in a bioreactor) issubsequently maintained in serum-free culture medium, wherein the mediumis a feed medium (for example eRDF medium), at a temperature lower thanthe growth temperature, which is suitable for protein expression andproduction of the secreted protein product (for example, aCTLA4^(A29YL104E)-Ig).

The commercial-scale culture is lowered, for example, from about 37° C.to about 34° C. for about 4 days. Polyanionic compound may be addedconcomitantly to the culture when the temperature is lowered.Alternatively, the commercial-scale culture is lowered from about 35°C-37° C. to about 32° C.-36° C. for about 12 days and polyanioniccompound is concomitantly added to the culture as the temperature islowered.

The feed medium in the instances described above can be replaced daily(1, 2, 3, etc. times daily) or every few days with fresh feed medium.The tank's replacement with fresh feed medium entails removal of apredetermined volume, for example 80% of the tank volume, and replacingthe tank with the same volume of fresh feed medium. In one embodiment,the feed medium is added daily for about 2 to 3 days until, for example,the glucose concentration falls to 1 g/L. In another embodiment, thefeed medium is added every 8 hours, for example, once the glucoseconcentration has reached 1 g/L. The commercial scale is maintaineduntil said CHO cells and/or secreted protein product reach a targetvalue of the following non-limiting production parameters: a NANA molarratio of about 6.0, or from about 5.2 to about 7.6; a final cell cultureviability ≥37%; and a final protein product titer of from about 0.46 toabout 0.71 g/L.

In an embodiment of the present invention, the cells being cultivatedcan be mammalian cells, or an established mammalian cell line,including, without limitation, CHO (e.g., ATCC CCL 61), HEK293 (e.g.,ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977), COS-1(e.g., ATCC CRL 1650), DG44 (CHO cell line) (Cell, 33: 405, 1983, andSomatic Cell and Molecular Genetics 12: 555, 1986), and baby hamsterkidney (BHK) cell lines. Other useful non-limiting examples aremyelomas, 3T3 cells, Namalwa cells, and fusions of myelomas with othercells. In some embodiments, the cells can be mutant or recombinantcells, such as, for example, cells that express a different spectrum ofenzymes that catalyze post-translational modification of proteins (e.g.,processing enzymes such as propeptides or glycosylation enzymes such asglycosyl transferases and/or glycosidases) than the cell type from whichthey were derived. In one particular aspect of this invention, CHOIdhfr-cells particularly are utilized.

The culturing vessels used for expanding the cell culture can be, butare not limited to, Erlenmyer flasks, T-175 flasks, roller bottles, andcell bags. The large-scale culture vessels can be, for example airliftreactors where agitation is obtained by means of introducing air fromthe bottom of the vessel or conventional stirred tank reactors (CSTR),where agitation is obtained by means of conventional impeller types.Among the parameters controlled within specified limits are temperature,pH, and dissolved oxygen tension (DOT). The temperature-control mediumin this system is water, and can be heated or cooled as necessary. Thewater can be passed through a piping coil immersed in the cell culturemedium or through a jacket surrounding the vessel. The pH, for example,can be regulated by addition of base to the cell culture medium whenrequired or by varying the CO₂ concentration in the head-space gas. DOTcan be maintained by sparging with pure oxygen or air or mixturesthereof

The invention therefore provides a method for producing a recombinantprotein, the method comprising at least two steps: (a) expandingmammalian cells that secrete a recombinant protein (i.e., a protein thatthe mammalian cells do not normally express or over-express, where therecombinant protein is expressed in the cells via an expression vectoror construct that has been transfected into the cells or the parents ofthe cells) from a seed culture to a liquid culture of at least 10,000 L,and (b) isolating the recombinant protein from the at least 10,000 Lliquid culture. In one embodiment, this method can be used such that therecombinant protein is produced at a concentration of at least 0.5 gramsper liter of liquid culture prior to purification of the protein fromthe liquid culture. In another embodiment, the method according to theinvention can be used to produce a recombinant protein at aconcentration of at least from about 0.46 to about 0.71 grams per literof liquid culture prior to purification of the protein from the liquidculture.

In one embodiment, the expansion step can involve (i) culturing thecells in a serum-free medium with at least four passages so as to obtaina cell density of at least about 1.0×10⁵ viable cells per mL, and (ii)maintaining the cells in culture for a time sufficient to produce atleast about 0.5 of the recombinant protein. In one embodiment, thenumber of passages does not exceed 36 passages. In another embodiment,the number of passages can exceed 36 passages where the cells are stableover generations with respect to copy number of the nucleic acid codingfor the recombinant protein, cell viability, and doubling time.

The time sufficient to produce at least about 0.5 to about 1.3 g/L ofthe recombinant protein can be any amount of time as long as the cellviability does not fall below 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%,90%, 95%, 98% and/or as long as the number of cell generations does notexceed 50, 75, 100, 105, or 125 generations. The maintaining step canalso comprise temperature shift steps, such as lowering the temperatureof the culture first from 37±2° C. to 34±2° C. and at a later time from34±2° C. to 32±2° C. The temperature of 32±2° C. can be maintained forat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 50,or 100 days. The temperature of 32±2° C. can be maintained for at least20, 50, 75, or 100 cell generations. The temperature of 32±2° C. can bemaintained until the cell density of the culture is from about 30 toabout 100×10⁵ cells per mL of liquid culture.

In other embodiments, the invention provides methods for producing arecombinant protein, the method comprising at least the steps of: (a)expanding mammalian cells that secrete a recombinant protein from a seedculture to a liquid culture of at least 10,000 L so that the recombinantprotein concentration is at least 0.5 grams/L of liquid culture; and (b)isolating the recombinant protein from the at least 10,000 L liquidculture when the liquid culture: (i) contains greater than or equal toabout 6.0 moles of NANA per mole of protein (glycoprotein in this case);(ii) has a cell density of from about 33 to about 79×10⁵ cells per mL;(iii) cell viability in the liquid culture is not less than about 38% oris greater than or equal to about 38%; (iv) endotoxin is less than orequal to about 76.8 EU per mL of liquid culture; and/or (v) bioburden isless than 1 colony forming unit per mL of liquid culture.

In a further embodiment, the expansion step can involve (i) culturingthe cells in a serum-free medium with at least four passages so as toobtain a cell density of at least about 1.0×10⁶ viable cells per mL, and(ii) maintaining the cells in culture for a time sufficient to produceat least from about 0.46 to about 0.71 grams of the recombinant proteinper literof liquid culture. In one embodiment, the number of passagesdoes not exceed 36 passages. In another embodiment, the number ofpassages can exceed 36 passages where the cells are stable overgenerations with respect to copy number of the nucleic acid coding forthe recombinant protein, cell viability, and doubling time.

The time sufficient to produce at from about 0.46 to about 0.71 g/L ofthe recombinant protein can be any amount of time as long as the cellviability does not fall below 5%, 10%, 25%, 30%, 50%, 60%, 70%, 80%,90%, 95%, 98% and/or as long as the number of cell generations does notexceed 27, 50, 75, 100, 105, or 125 generations. The maintaining stepcan also comprise temperature shift steps, such as lowering thetemperature of the culture first from 37±2° C. to 34±2° C. Thetemperature of 34±2° C. can be maintained for at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 50, or 100 days.Alternatively, the maintaining step can a temperature shift step, suchas lowering the temperature of the culture from 37±2° C. to 34±2° C.

Polyanionic compound can be added to the cultures as temperaturelowering commences. The concentration of polyanionic compound added tothe culture can be about 1 mg/L, 5 mg/L, 10 mg/L, 12.5 mg/L, 15 mg/L, 25mg/L, 50 mg/L, 75 mg/L, 100 mg/L, 200 mg/L, 250 mg/L, 500 mg/L, 750mg/L, or 1000 mg/L. The temperature of 32±2° C. can be maintained for atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 30, 50, or100 days. The temperature of 34±2° C. can be maintained for at least 20,27, 50, 75, or 100 cell generations.

In further embodiments, the invention provides methods for producing arecombinant protein, the method comprising at least the steps of: (a)expanding mammalian cells that secrete a recombinant protein from a seedculture to a liquid culture of at least 10,000 L so that the recombinantprotein concentration is at least from about 0.46 to about 0.71 gramsper liter of liquid culture; and (b) isolating the recombinant proteinfrom the culture of at least 10,000 L liquid culture when the liquidculture: (i) contains about 6 moles of NANA per mole of protein(glycoprotein in this case); (ii) cell viability in the liquid cultureis not less than about 37%; (iii) endotoxin is less than or equal toabout 4.8 EU per mL of liquid culture; and/or (iv) bioburden is lessthan 1 colony forming unit per mL of liquid culture.

The recombinant protein produced by these methods of the invention canbe a secreted protein, a glycoprotein, a cytokine, a hormone, a CTLA4-Igprotein, or a CTLA4^(A29YL104E)-Ig protein. In one embodiment, themammalian cells are progeny or subclones of cells provided by theinvention. In another embodiment, the mammalian cells are progeny orsubclones of cells derived from the cell line of the invention. In afurther embodiment, the mammalian cells are a clonal population fromcells transfected with an expression cassette comprising SEQ ID NO:1. Ina particular embodiment, the mammalian cells are a clonal populationfrom cells transfected with an expression cassette comprising SEQ IDNO:3.

General Techniques for the Purification of Recombinant Protein fromCulture

Following the protein production phase of the cell culture process, theprotein of interest, for example a glycoprotein, is recovered from thecell culture medium using techniques understood by one skilled in theart. In particular, the protein of interest is recovered from theculture medium as a secreted polypeptide, although it also can berecovered from host cell lysates. The culture medium or lysate isinitially centrifuged to remove cellular debris and particulates. Thedesired protein subsequently is purified from contaminant DNA, solubleproteins, and polypeptides, with the following non-limiting purificationprocedures well-established in the art: SDS-PAGE; ammonium sulfateprecipitation; ethanol precipitation; fractionation on immunoaffinity orion-exchange columns; reverse phase HPLC; chromatography on silica or onan anion-exchange resin such as QAE or DEAE; chromatofocusing; gelfiltration using, for example, Sephadex G-75™ column; and protein ASepharose™ columns to remove contaminants such as IgG. Addition of aprotease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF), or aprotease inhibitor cocktail mix also can be useful to inhibitproteolytic degradation during purification. A person skilled in the artwill recognize that purification methods suitable for a protein ofinterest, for example a glycoprotein, can require alterations to accountfor changes in the character of the protein upon expression inrecombinant cell culture.

Purification techniques and methods that select for the carbohydrategroups of the glycoprotein are also of utility within the context of thepresent invention. For example, such techniques include, HPLC orion-exchange chromatography using cation-or anion-exchange resins,wherein the more basic or more acidic fraction is collected, dependingon which carbohydrate is being selected for. Use of such techniques alsocan result in the concomitant removal of contaminants.

In the present invention, CHO cells capable of producing CTLA4-Ig orCTLA4^(A29YL104E)-Ig fusion proteins are grown as a suspension in a CHOspecific medium to a predetermined cell density. CHO cells grown insuspension in the serum-free expression medium subsequently produceCTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules, which are secreted by theCHO cells into the culture medium. The cell suspension can be clearedvia centrifugation and CTLA4-Ig molecules can then be separated from thecleared culture supernatant by standard purification techniques.Non-limiting examples of suitable purification procedures for obtaininggreater purity and homogeneity of CTLA4-Ig or CTLA4^(A29YL104E)-Ig,either individually or in combination, are: affinity chromatography onsepharose; fractionation on anion-exchange columns (AEC); andhydrophobic interaction chromatography (HIC).

In some embodiments, isolating CTLA4-Ig molecules or other proteins(including glycoproteins) from the methods of production describedherein can at least include the following steps: (i) obtaining a cellculture supernatant; (ii) subjecting the supernatant to anion exchangechromatography to obtain an eluted protein product; (iii) subjecting theeluted protein product of step (ii) to hydrophobic interactionchromatography so as to obtain an enriched protein product; (iv)subjecting the enriched protein product to affinity chromatography toobtain an eluted and enriched protein product; and (v) subjecting theeluted and enriched protein product of (iv) to anion exchangechromatography. The enriched protein product obtained in step (iii) canbe charactertized, for example, in that its percentage of any HMWprotein or contaminant is less than 5, 10, 15 or 25%. The anion exchangechromatography of step (ii) can be carried out, for example, by using awash buffer comprising about 25-100 mM HEPES and about 300-900 mM NaCland having a pH of about 7.0-8.0. The hydrophobic interactionchromatography of step (iii) can be carried out, for example, by using asingle wash buffer having a pH of about 7.0 and comprising about 25 mMHEPES and about 850 mM NaCl; or a wash buffer having a pH of about 8.0and comprising about 25 mM Tris and about 250 mM NaCl. The affinitychromatography of step (iv) can be carried out, for example, by using anelution buffer having a pH of about 3.5 and comprising about 100 mMglycine. The affinity chromatography of step (v) can be carried out, forexample, by using a wash buffer having a pH of about 8.0 and comprisingabout 25 mM HEPES and from about 120 mM NaCl to about 130 mM NaCl, or awash buffer having a pH of about 8.0 and comprising about 25 mM HEPESand about 200 mM NaCl. The anion exchange chromatography of step (ii)can be carried out using a column having an anion exchange resin havinga primary, secondary, tertiary, or quartenary amine functional group.The hydrophobic interaction column of step (iii) can be carried outusing a hydrophobic interaction resin having a phenyl, an octyl, apropyl, an alkoxy, a butyl, or an isoamyl functional group.

In other embodiments, isolating CTLA4^(A29YL104E)-Ig molecules or otherproteins (including glycoproteins) from the methods of productiondescribed herein can at least include the following steps: (i) obtaininga cell culture supernatant; (ii) subjecting the supernatant to affinitychromatography to obtain an eluted protein product; (iii) subjecting theeluted protein product of step (ii) to anion exchange chromatography soas to obtain an enriched protein product; and (iv) subjecting theenriched protein product to hydrophobic interaction chromatography toobtain an eluted and enriched protein product with reduced highmolecular weight (HMW) protein complexes. The enriched protein productobtained in step (iv) can be characterized, for example, in that itspercentage of any HMW protein or contaminant is less than 5, 10, 15 or25%. The affinity chromatography of step (ii) can be carried out, forexample, by using an elution buffer having a pH of about 3.0 andcomprising about 250 mM glycine. The affinity chromatography of step(ii) can be carried out, for example, by using a wash buffer having a pHof about 7.5 and comprising about 25 mM NaH₂PO₄ and about 150 mM NaCl.The anion exchange chromatography of step (iii) can be carried out, forexample, by using a wash buffer comprising about 50 mM HEPES and about135 mM NaCl and having a pH of about 7.0. The anion exchangechromatography of step (iii) can be carried out, for example, by usingan elution buffer comprising about 50 mM HEPES and about 200 mM NaCl andhaving a pH of about 7.0. The hydrophobic interaction chromatography ofstep (iv) can be carried out, for example, by using a wash buffer havinga pH of about 7.0 and comprising about 50 mM HEPES and about 1.2 M(NH₄)₂SO₄. The anion exchange chromatography of step (iii) can becarried out using a column having an anion exchange resin having aprimary, secondary, tertiary, or quartenary amine functional group. Thehydrophobic interaction column of step (iv) can be carried out using ahydrophobic interaction resin having a phenyl, an octyl, a propyl, analkoxy, a butyl, or an isoamyl functional group.

In one embodiment, the invention provides a method for purifyingCTLA4-Ig molecules from a liquid cell culture so that the purifiedCTLA4-Ig is substantially free of Monocyte Chemotactic Protein-1(MCP-1). In one embodiment, the invention provides for apharmaceutically acceptable composition of CTLA4-Ig molecules, whereinthe composition comprises no more than 0.5 ppm MCP-1, 1 ppm MCP-1, 2 ppmMCP-1, 3 ppm MCP-1, 4 ppm MCP-1, 5 ppm MCP-1, 6 ppm MCP-1, 7 ppm MCP-1,8 ppm MCP-1, 9 ppm MCP-1 or 10 ppm MCP-1. In another embodiment, in thecomposition, the amount of MCP-1 cannot exceed 1%, 0.5%, or 0.1% of theweight of purified CTLA4-Ig. In another embodiment, the composition ofCTLA4-Ig molecules is substantially free of MCP-1 where there is lessthan 50, 45, 40, 38, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or1 ng/mL of MCP-1 in the QFF eluate liquid. In another embodiment, theinvention provides a method for purifying CTLA4-Ig molecules from aliquid cell culture so that the purified CTLA4-Ig is substantially freeof MCP-1 and comprises less than 2.5% of CTLA4-Ig tetramer.

The amount of Monocyte chemotactic protein 1 (MCP-1) in the compositioncan be quantified using an ELISA method. The coating antibody is a goatanti-mouse MCP-1 IgG antibody. The secondary antibody is a rabbitanti-rat MCP-1 IgG antibody. Detection is accomplished using horseradishperoxidase conjugated goat anti-rabbit IgG antibody and the substrateTMB. The horseradish peroxidase reagent produces a colorimetric reactionthat develops in proportion to the amount of protein captured. The ELISAquantifies the MCP-1 level relative to a material standard curve. In oneembodiment, MCP-1 was quantified in the composition and the MCP-1 levelswere in ranges from 0-0.097 ng/mg and 0.014 0.154 ng/mg.

In another embodiment, the invention provides a method for purifyingCTLA4^(A29YL104E)-Ig molecules from a liquid cell culture so that thepurified CTLA4^(A29YL104E)-Ig is substantially free of MonocyteChemotactic Protein-1 (MCP-1). In one embodiment, the amount of MCP-1cannot exceed 1%, 0.5%, or 0.1% of the weight of purifiedCTLA4^(A29YL104E)-Ig. In another embodiment, CTLA4^(A29YL104E)-Ig issubstantially free of MCP-1 where there is less than 50, 45, 40, 38, 35,or 30 ng/mL of MCP-1 in the HIC eluate liquid. In a further embodiment,the invention provides a method for purifying CTLA4^(A29YL104E)-Igmolecules from a liquid cell culture so that the purifiedCTLA4^(A29YL104E)-Ig is substantially free of MCP-1 and comprises lessthan 2.5% of CTLA4^(A29YL104E)-Ig tetramer.

Glycoprotein Recovery from the Cell Culture and Purification

The present invention describes a series of steps for the separation ofa glycoprotein (for example, CTLA4-Ig or CTLA4^(A29YL104E)-Ig) from animpure, cell-culture supernatant, protein pool that contains theglycoprotein of interest (such as CTLA4-Ig or CTLA4^(A29YL104E)-Ig) andundesirable contaminants. The impure, cell-culture supernatant can beused as the starting material for the purification of the CTLA4-Ig orCTLA4^(A29YL104E)-Ig glycoprotein.

In one embodiment of the present invention, the impure, cell-culturesupernatant that contains CTLA4-Ig glycoprotein and undesirablecontaminants is applied to an anion-exchange medium. The CTLA4-Igglycoprotein present in the impure, cell-culture supernatant binds tothe anion-exchange medium. The anion-exchange medium is then washed toremove any unbound material from the anion-exchange medium. CTLA4-Igglycoprotein is eluted after the unbound material is removed, and theeluate is collected.

In one embodiment of the present invention, the impure, cell-culturesupernatant that contains CTLA4^(A29YL104E)-Ig glycoprotein andundesirable contaminants is applied to an affinity chromatographymedium. The CTLA4^(A29YL104E)-Ig glycoprotein present in the impure,cell-culture supernatant binds to the affinity chromatography medium.The affinity chromatography medium is then washed to remove any unboundmaterial from the anion-exchange medium. CTLA4^(A29YL104E)-Igglycoprotein is eluted after the unbound material is removed, and theeluate is collected.

In a particular embodiment of this invention, Q-Sepharose Anion-ExchangeChromatography (AEC), for example using a Q-Sepharose XL column (GEHealthcare), is employed to separate CTLA4-Ig glycoprotein from theharvest material, as well as for decreasing bulk contaminants. Thiscolumn can be used as an early step in the purification of CTLA4-Igglycoprotein from a mammalian cell culture, for fractionation of theharvested cell culture medium. In another embodiment, Q-SepharoseAnion-Exchange Chromatography, for example Q-Sepharose Fast Flow (GEHealthcare), can be used after an affinity chromatography purificationstep. The very high flow property of the anion exchange columns allowsthe large volume of CTLA4-Ig glycoprotein or harvested cell culturemedium to be readily concentrated before subsequent chromatographysteps, such as SP-Sepharose or HIC, by adjusting conditions so that theCTLA4-Ig glycoprotein binds the column. For a wash buffer of pH fromabout pH 5 to 9, in particular about 8, 75 mM HEPES and 360 mM NaClconcentrations are useful. Typically, for an elution buffer of pH fromabout pH 5 to 8, in particular about 7, 25 mM HEPES and 325 mM NaClconcentrations are useful.

Suitable resins for separating CTLA4-Ig glycoprotein from the harvestedculture medium were those having immobilized amine functional groups.Most useful are the quarternary amine functional group resins, forexample those on Q-Sepharose Fast Flow resins from GE Healthcare, wherea quarternary ammonium ligand is bound to high-porosity, cross-linkedagarose. Also useful are the primary, secondary and tertiary aminefunctional group resins, for example those on DEAE Sepharose Fast Flowresins from GE Healthcare, where a tertiary diethylaminoethyl ligand isbound to high-porosity, cross-linked agarose.

In another embodiment of the present invention, the CTLA4-Igglycoprotein-containing eluate from the anion-exchange medium iscollected and then contacted with a hydrophobic interaction resin. Asdescribed below, the CTLA4-Ig glycoprotein-containing volume passesthrough the HIC column and the collected pool, which can be furtherpurified, is then bound to an anion-exchange resin.

HIC is useful for the separation of desired CTLA4-Ig glycoprotein dimersfrom high molecular weight material and other protein impurities fromthe mammalian cell culture. For example, CTLA4-Ig-expressing-CHO cellculture contains high molecular weight aggregates of CTLA4-Igglycoprotein. Also found in the mammalian cell culture medium are CHOcell protein impurities. These undesirable products could generate anunwanted antigenic response in a patient and contribute to poor productquality or activity. HIC effectively separates hydrophobic variants,CTLA4-Ig glycoprotein dimers from CTLA4-Ig glycoprotein HMW complexesand CHO protein impurities via the latter products binding to the HICresin and the CTLA4-Ig glycoprotein dimers passing through the column.Thus, a CTLA4-Ig glycoprotein pool could be obtained that issubstantially free of these species, and that is particularly suited foranother chromatographic step, such as anion-exchange chromatography. Asource of CTLA4-Ig glycoprotein mixtures for use with HIC is mammaliancell culture, for example a CHO cell culture. In particular, the culturecan be subjected to at least one prior purification step as discussedpreviously.

In another embodiment of this invention, the HIC method can be modifiedto collect a pool of other glycoproteins (for example, CTLA4-Ig HMWcomplexes). HMW aggregates can bind to the HIC resin (for example,comprising CTLA4-Ig tetramer and the like). These HMW complexes have ahigher avidity and bind more efficaciously in vivo than CTLA4-Ig dimeralone. Thus, one skilled in the art can obtain a pool of CTLA4-Ig HMWaggregates by eluting the CTLA4-Ig pool off of the HIC.

The most useful HIC resins for separating CTLA4-Ig glycoprotein formsare those having immobilized phenyl functional groups. Of the phenyl-HICresins, Phenyl Sepharose Fast Flow High Sub (high substitution) by GEHealthcare is most useful. Phenyl Toyopearl media by TosoHaas and TSKPhenyl 5PW are non-limiting examples of other phenyl-HIC resins that canbe used. Other HIC functional groups include, but are not limited to,the propyl, octyl, alkoxyl, butyl, and isoamyl moieties.

For example, a Phenyl Sepharose 4 Fast Flow column chromatography,Hydrophobic Interaction Chromatography (HIC), process can be used toreduce the amount of CTLA4-Ig or CTLA4^(A29YL104E)-Ig high molecularweight species eluted in a HIC purification step (see Example 15 andEXAMPLE 20). Therefore, the cleaning peak from the HIC column isenriched in CTLA4-Ig or CTLA4^(A29YL104E)-Ig HMW species.

The unbound fraction containing CTLA4-Ig glycoprotein from the HICpurification step can be subjected to an additional purification method,such as affinity chromatography, and the resulting eluate can then beapplied to an anion-exchange medium. The CTLA4-Ig glycoprotein binds tothe anion-exchange resin, which can be subsequently washed to removeunbound proteins. After the unbound proteins are removed, CTLA4-Igglycoprotein is eluted from the second anion-exchange resin. The eluateis collected and can be further concentrated.

In another embodiment of this invention, affinity chromatography, forexample rProtein A Sepharose Fast Flow (GE Healthcare), is employed tofurther enrich CTLA4-Ig glycoprotein, which can be further followed byan anion-exchange chromatography step, for example Q-Sepharose Fast Flow(GE Healthcare). The affinity chromatography step can also reduce thelevels of CHO proteins and Monocyte Chemotactic Protein (MCP-1, achemokine) impurities. Affinity chromatography involves adsorptiveseparation, where a molecule of interest to be purified, for exampleCTLA4-Ig glycoprotein, binds specifically and reversibly to a ligandimmobilized on some matrix or resin. Some non-limiting examples ofaffinity purification columns include lectin; affinity tag (for example,a GST column or 6X-His column); Streptavidin; heparin; or antibody (forexample, a Protein A column or a Protein G column). In particular, thisinvention utilizes a protein A resin for binding the CTLA4-Igglycoprotein. For a wash buffer of pH from about 5 to 9, more effectiveat about 8, 25 mM Tris and 250 mM NaCl concentrations are useful. For anelution buffer of pH from about 2 to 5, more effective at about 3.5, 100mM glycine concentration is useful. The affinity chromatography eluatecan then be neutralized and loaded onto an anion exchange chromatographycolumn, Q-Sepharose Fast Flow being most useful.

To further reduce the levels of protein A, DNA, and non-desired CTLA4-Igglycoprotein species in the product after the foregoing recovery/initialpurification steps, another ion-exchange step can be incorporated intothe purification procedure. This invention can employ commerciallyavailable ion-exchange columns, such as a Q-Sepharose Fast Flow columnfrom GE Healthcare, or a DEAE Sepharose Fast Flow column, also from GEHealthcare. As determined herein, the most suitable resins forseparating CTLA4-Ig glycoprotein from the harvested culture medium werethose having immobilized amine functional groups. Other useful groupsare the quarternary amine functional group resins, for example those ina Q-Sepharose Fast Flow column from GE Healthcare, where a quarternaryammonium ligand is bound to high-porosity, cross-linked agarose. Alsouseful are the primary, secondary and tertiary amine functional groupresins, for example those in a DEAE Sepharose Fast Flow column from GEHealthcare, where a tertiary diethylaminoethyl ligand is bound tohigh-porosity, cross-linked agarose. In a particular embodiment of theinvention, a column that utilizes a strong anion exchanger, such as aQ-Sepharose Fast Flow column, is utilized.

In one embodiment of the invention, a CTLA4-Ig glycoprotein eluate isloaded onto an anion exchange column, for example Q-Sepharose Fast Flow.The column is washed and CTLA4-Ig glycoprotein is subsequently elutedfrom the anion exchange column. For a wash buffer of pH from about 5 to9, in one embodiment, pH 8, 25 mM HEPES and 100-140 mM NaClconcentrations are useful. For an elution buffer of pH from about 5 to9, or in another embodiment, pH 8, 25 mM HEPES and 200 mM NaClconcentrations are useful. CTLA4-Ig glycoprotein eluted from theanion-exchange medium is recovered, concentrated and washed, bydiafiltration or other suitable method known to one skilled in the art,to provide a final purified CTLA4-Ig glycoprotein product. The CTLA4-Igglycoprotein product prepared in accordance with the process of thepresent invention is of high purity, for example containing ≥95% of theCTLA4-Ig dimer, containing ≤5% of the CTLA4-Ig HMW product, andcontaining ≤1% of CTLA4-Ig monomer.

The purification method can further comprise additional steps thatinactivate and/or remove viruses and/or retroviruses that mightpotentially be present in the cell culture medium of mammalian celllines. A significant number of viral clearance steps are available,including but not limited to, treating with chaotropes such as urea orguanidine, detergents, additional ultrafiltration/diafiltration steps,conventional separation, such as ion-exchange or size exclusionchromatography, pH extremes, heat, proteases, organic solvents or anycombination thereof

In another embodiment of this invention, affinity chromatography, forexample MabSelect Protein A Sepharose resin (GE Healthcare), is employedto capture CTLA4^(A29YL104E)-Ig glycoprotein, which can be furtherfollowed by an anion-exchange chromatography step, for exampleQ-Sepharose Fast Flow (GE Healthcare). The affinity chromatography stepcan also reduce the levels of CHO proteins and Monocyte ChemotacticProtein (MCP-1, a chemokine) impurities. Affinity chromatographyinvolves adsorptive separation, where a molecule of interest to bepurified, for example CTLA4^(A29YL104E)-Ig glycoprotein, bindsspecifically and reversibly to a ligand immobilized on some matrix orresin. Some non-limiting examples of affinity purification columnsinclude lectin; affinity tag (for example, a GST column or 6X-Hiscolumn); Streptavidin; heparin; or antibody (for example, a Protein Acolumn or a Protein G column). In particular, this invention utilizes aprotein A resin for binding the CTLA4^(A29YL104E)-Ig glycoprotein. For awash buffer of pH from about 5 to 9, more effective at about 7.5, 25 mMTris, 25 mM NaH₂PO₄, and 250 mM NaCl concentrations are useful. For anelution buffer of pH from about 2 to 5, more effective at about 3.5,100-300 mM glycine concentration is useful. The affinity chromatographyeluate can then be neutralized and loaded onto an anion exchangechromatography column, Q-Sepharose Fast Flow being most useful.

To further reduce the levels of protein A, DNA, and non-desiredCTLA4^(A29YL104E)-Ig glycoprotein species in the product after theforegoing recovery/initial purification steps, an ion-exchange step canbe incorporated into the purification procedure. This invention canemploy commercially available ion-exchange columns, such as aQ-Sepharose Fast Flow column from GE Healthcare, Q-Sepharose XL column(GE Healthcare), or a DEAE Sepharose Fast Flow column, also from GEHealthcare. The most suitable resins for separating CTLA4^(A29YL104E)-Igglycoprotein are those having immobilized amine functional groups. Otheruseful groups are the quarternary amine functional group resins, forexample those in a Q-Sepharose Fast Flow column from GE Healthcare,where a quarternary ammonium ligand is bound to high-porosity,cross-linked agarose. Also useful are the primary, secondary andtertiary amine functional group resins, for example those in a DEAESepharose Fast Flow column from GE Healthcare, where a tertiarydiethylaminoethyl ligand is bound to high-porosity, cross-linkedagarose. In a particular embodiment of the invention, a column thatutilizes a strong anion exchanger, such as a Q-Sepharose Fast Flowcolumn, is utilized.

In one embodiment of the invention, a CTLA4^(A29YL104E)-Ig glycoproteineluate is loaded onto an anion exchange column, for example Q-SepharoseFast Flow. The column is washed and CTLA4^(A29YL104E)-Ig glycoprotein issubsequently eluted from the anion exchange column. For a wash buffer ofpH from about 5 to 9, in one embodiment, pH 7, 25-55 mM HEPES and100-140 mM NaCl concentrations are useful. For an elution buffer of pHfrom about 5 to 9, or in another embodiment, pH 7, 25-50 mM HEPES and200 mM NaCl concentrations are useful.

In another embodiment of the present invention, the CTLA4^(A29YL104E)-Igglycoprotein-containing eluate from the anion-exchange medium iscollected and then contacted with a hydrophobic interaction resin. HICis useful for the separation of desired CTLA4^(A29YL104E)-Igglycoprotein dimers from high molecular weight material and otherprotein impurities from the mammalian cell culture. For example,CTLA4^(A29YL104E)-Ig-expressing-CHO cell culture contains high molecularweight aggregates of CTLA4^(A29YL104E)-Ig glycoprotein. Also found inthe mammalian cell culture medium are CHO cell protein impurities. Theseundesirable products could generate an unwanted antigenic response in apatient and contribute to poor product quality or activity.

HIC effectively separates hydrophobic variants, CTLA4^(A29YL104E)-Igglycoprotein dimers from CTLA4^(A29YL104E)-Ig glycoprotein HMW complexesand CHO protein impurities via the latter products binding to the HICresin and the CTLA4^(A29YL104E)-Ig glycoprotein dimers passing throughthe column. Thus, a CTLA4^(A29YL104E)-Ig glycoprotein pool could beobtained that is substantially free of these species. A source ofCTLA4^(A29YL104E)-Ig glycoprotein mixtures for use with HIC is mammaliancell culture, for example a CHO cell culture. In particular, the culturecan be subjected to at least one prior purification step as discussedpreviously.

In another embodiment of this invention, the HIC method can be modifiedto collect a pool of other glycoproteins (for example,CTLA4^(A29YL104E)-Ig HMW complexes). HMW aggregates can bind to the HICresin (for example, comprising CTLA4^(A29YL104E)-Ig tetramer and thelike). These HMW complexes have a higher avidity and bind moreefficaciously in vivo than CTLA4^(A29YL104E)-Ig dimer alone. Thus, oneskilled in the art can obtain a pool of CTLA4^(A29YL104E)-Ig HMWaggregates by eluting the CTLA4^(A29YL104E)-Ig pool off of the HIC.

CTLA4^(A29YL104E)-Ig glycoprotein eluted from the HIC medium isrecovered, concentrated and washed, by diafiltration or other suitablemethod known to one skilled in the art, to provide a final purifiedCTLA4^(A29YL104E)-Ig glycoprotein product. The CTLA4^(A29YL104E)-Igglycoprotein product prepared in accordance with the process of thepresent invention is of high purity, for example containing >95% of theCTLA4^(A29YL104E)-Ig dimer, containing <5% of the CTLA4^(A29YL104E)-IgHMW product, and containing ≤1% of CTLA4^(A29YL104E)-Ig monomer.

The most useful HIC resins for separating CTLA4^(A29YL104E)-Igglycoprotein forms are those having immobilized phenyl functionalgroups. Of the phenyl-HIC resins, Phenyl Sepharose Fast Flow High Sub(high substitution) by GE Healthcare is most useful. Phenyl Toyopearlmedia by TosoHaas and TSK Phenyl 5PW are non-limiting examples of otherphenyl-HIC resins that can be used. Other HIC functional groups include,but are not limited to, the propyl, octyl, alkoxyl, butyl, and isoamylmoieties.

The purification method can further comprise additional steps thatinactivate and/or remove viruses and/or retroviruses that mightpotentially be present in the cell culture medium of mammalian celllines. A significant number of viral clearance steps are available,including but not limited to, treating with chaotropes such as urea orguanidine, detergents, additional ultrafiltration/diafiltration steps,conventional separation, such as ion-exchange or size exclusionchromatography, pH extremes, heat, proteases, organic solvents or anycombination thereof.

In one aspect, purified CTLA4-Ig molecules which have been concentratedand subjected to diafiltration step can be filled into 2-L BIOTAINER®bottles, 50-L bioprocess bag or any other suitable vessel. CTLA4-Igmolecules in such vessels can be stored for about 60 days at 2° to 8° C.prior to freezing. Extended storage of purified CTLA4-Ig at 2° to 8° C.may lead to an increase in the proportion of CLTA4-Ig tetramer.Therefore, for long-term storage, CTLA4-Ig molecules can be frozen atabout −70° C. prior to storage and stored at a temperate of about −40°C. The freezing temperature can vary from about −50° C. to about −90° C.The freezing time can vary and largely depends on the volume of thevessel that contains CTLA4-Ig molecules, and the number of vessels thatare loaded in the freezer. For example, in one embodiment, CTLA4-Igmolecules are in 2-L BIOTAINER® bottles. Loading of less than four 2-LBIOTAINER® bottles in the freezer may require from about 14 to at least18 hours of freezing time. Loading of at least four bottles may requirefrom about 18 to at least 24 hours of freezing time. Vessels with frozenCTLA4-Ig molecules are stored at a temperature from about −35° C. toabout −55° C.

The storage time at a temperature of about −35° C. to about −55° C. canvary and can be as short as 18hours. The frozen CTLA4-Ig molecules canbe thawed in a control manner. Thawing of frozen CTLA4-Ig molecules iscontrolled and can be done in an incubator at a temperature from about20° C. to about 24° C. The duration of the thawing steps depends on theloading of the incubator wherein loading of less than four 2-L BIOTAINER® bottles may require less than about 24 hours of thawing time. Loadingof four 2-L BIOTAINER bottles may require about 18 hours. Thawedsolution comprising CTLA4-Ig molecules can be mixed to avoid potentialconcentration gradients. Therefore, thawing can be done in acontrolled-temperature incubator, which also allows for shaking of thevessels, which contain CTLA4-Ig. The speed of shaking can be from about40 to about 80 rpm. Thawed CTLA4-Ig molecules can be further mixed foradditional 5-10 min at a rotational rate of about 3 rpm. Thawed CTLA4-Igmolecules can be stored at 2° to 8° C., alequated and lyophilized duringthe production of pharmaceutical compositions comprising CTLA4-Ig.

The present invention can be further applied to the purification ofother, non-limiting examples, of therapeutic glycoproteins produced inlarge scale. The process of this invention can be applicable to theproduction of other glycoproteins having more than one glycosylatedvariant in mammalian cell cultures. One skilled in the art willunderstand the modifications that might become necessary in the courseof the adaptation of the exemplified method to the production ofdifferent glycoproteins.

Formulations & Kits

The invention also provides any of the described CTLA4-Ig molecules as alyophilized mixture. Formulations comprising CTLA4-Ig to be lyophilizedcan further comprise three basic components: (1) an additional activeingredient(s) including other recombinant proteins or small molecules(such as immunosuppressants), (2) an excipient(s) and (3) a solvent(s).Excipients include pharmaceutically acceptable reagents to provide goodlyophilized cake properties (bulking agents) as well as to providelyoprotection and/or cryoprotection of proteins (“stabilizer”),maintenance of pH (buffering agents), and proper conformation of theprotein during storage so that substantial retention of biologicalactivity (including active ingredient stability, such as proteinstability) is maintained. With respect to excipients, an example of aformulation can include one or more of a buffering agent(s), a bulkingagent(s), a protein stabilizer(s) and an antimicrobial(s). Sugars orpolyols can be used as nonspecific protein stabilizers in solution andduring freeze-thawing and freeze-drying. Polymers can be used tostabilize proteins in solution and during freeze-thawing andfreeze-drying. One popular polymer is serum albumin, which has been usedboth as a cryoprotectant and lyoprotectant. In one embodiment, theinvention provides formulations that are albumin free. Various salts canbe used as bulking agents. Illustrative salt bulking agents include, forexample, NaCl, MgCl₂ and CaCl₂. Certain amino acids can be used ascryoprotectants and/or lyoprotectants and/or bulking agents. Amino acidsthat can be used include, but are not limited to, glycine, proline,4-hydroxyproline, L-serine, sodium glutamate, alanine, arginine andlysine hydrochloride. Many buffering agents covering a wide pH range areavailable for selection in formulations. Buffering agents include, forexample, acetate, citrate, glycine, histidine, phosphate (sodium orpotassium), diethanolamine and Tris. Buffering agents encompasses thoseagents which maintain the solution pH in an acceptable range prior tolyophilization. Formulations have previously been described in U.S.Patent Application No. 60/752,150, filed Dec. 20, 2005, which is herebyincorporated by reference in its entirety.

In one embodiment, the invention provides a lyophilized CTLA4-Ig mixturecomprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimer. In oneembodiment, the invention provides a lyophilized CTLA4-Ig mixturecomprising at least 90%, 95%, 99%, or 99.5% CTLA4-Ig dimer and not morethan 5%, 4%, 3%, 2%, or 1% CTLA4-Ig tetramer. In another embodiment, theinvention provides a lyophilized CTLA4-Ig mixture comprising at least90%, 95%, 99%, or 99.5% CTLA4-Ig dimer, and not more than 5%, 4%, 3%,2%, or 1% CTLA4-Ig tetramer, and not more than 2%, 1.5%, 1.0%, 0.8%,0.5%, or 0.3% CTLA4-Ig monomer. In a further embodiment, the inventionprovides a lyophilized CTLA4-Ig mixture comprising at least 8.0 moles ofsialic acid per mole of CTLA4-Ig dimer or to CTLA4-Ig molecule. Inanother embodiment, the invention provides a lyophilized CTLA4-Igmixture comprising: from about 15 to about 35 moles of GlcNac per moleof CTLAIg molecules or dimer; from about 1 to about 5 moles of GalNacper mole of CTLA4-Ig dimer or to CTLA4-Ig molecule; from about 5 molesto about 20 moles of galactose per mole of CTLA4-Ig dimer or to CTLA4-Igmolecule; from about 2 to about 10 moles of fucose per mole of CTLA4-Igdimer or to CTLA4-Ig molecule; and/or from about 5-15 moles of mannoseper mole of CTLA4-Ig dimer or to CTLA4-Ig molecule

A CTLA4^(A29YL104E)-Ig drug substance is available as an aqueoussolution at approximately 25 mg/mL (22.5-27.5 mg/mL) concentration in 25mM sodium phosphate and 10 mM sodium chloride buffer at pH ˜7.5.CTLA4^(A29YL104E)-Ig has a tendency to form high molecular weightspecies in aqueous solution. Therefore, a freeze-dried product wasdeveloped in order to minimize the levels of high molecular weightspecies that may form in the drug product. Various excipients such asmaltose, sucrose, and amino acids such as L-arginine hydrochloride werescreened as potential lyoprotectants during freeze drying ofCTLA4^(A29YL104E)-Ig. Sucrose was found to be the most effectivelyoprotectant. It was further observed that increasing the sucrose toprotein ratio improved protein stability. A sucrose: protein ratio of2:1 (wt.:wt.) was chosen for the protein solution to be freeze dried.The freeze-dried drug product has adequate stability and satisfactoryconstitution behavior.

Methods of Treatment

According to this invention, a disease mediated by T cell interactionswith B7 positive cells can be treated by receiving a pharmaceuticallyacceptable formulation of CTLA4-Ig or CTLA4^(A29YL104E)-Ig. The CTLA4-Igor CTLA4^(A29YL104E)-Ig molecules secreted by an engineered mammaliancell line (for example, a dhf r-negative Chinese Hamster Ovary cell linethat harbors DNA encoding CTLA4-Ig or CTLA4^(A29YL104E)-Ig) can be apopulation of molecules having a particular glycosylation profile. Asstated herein, a particular glycoyslation profile can affect CTLA4-Ig orCTLA4^(A29YL104E)-Ig binding to CD80 and/or CD86 such that CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules can provide a greater inhibition on Tcell activation and/or proliferation. As stated herein, a particularglycosylation profile can be affected by the cell line and the method ofproduction. Thus, in certain embodiments of the invention, the inventionprovides CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules produced by a cellline in a production method described herein in order to treat T-cellrelated diseases or disorders, that include but are not limited to,generally any T-cell dependent lymphoproliferative disease or disorderand any T-cell dependent autoimmune disease or disorder, and morespecifically: T cell lymphoma, T cell acute lymphoblastic leukemia,testicular angiocentric T cell lymphoma, benign lymphocytic angiitis,graft versus host disease (GVHD), immune disorders associated with grafttransplantation rejection, psoriasis, inflammation, allergy, oophoritis,inflammatory bowel disease, glomerulonephritis, encephalomyelitis,Hashimoto's thyroiditis, Graves' disease, Addison's disease, Crohn'sdisease, Sjogren's syndrome, lupus erythematosus, primary myxedema,pernicious anemia, autoimmune atrophic gastritis, rheumatoid arthritis,insulin dependent diabetes mellitis, good pasture's syndrome, myastheniagravis, pemphigus, multiple sclerosis, sympathetic ophthalmia,autoimmune uveitis, autoimmune hemolytic anemia, idiopathicthrombocytopenia, primary biliary cirrhosis, chronic action hepatitis,ulceratis colitis, scleroderma, polymyositis, and mixed connectivetissue disease.

The invention provides the use of any of the disclosed CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules in methods for inhibiting T cellproliferation or activation, inhibiting an immune response in a subjector in vitro, and for treating an immune disorder in a subject orinducing immune tolerance to an antigen in a subject. Immune toleranceis a type of immunological response in which there develops a specificnonreactivity of the lymphoid tissues towards a specific antigen, wherein the absence of tolerance, the antigen is able to induce an immuneresponse. In one embodiment, the CTLA4-Ig or CTLA4^(A29YL104E)-Igmolecules and compositions of the invention can be used to treat asubject who has received a transplant in order to induce tolerance, andreduce the possibility of rejection. In another embodiment, thetransplant is an organ transplant, a tissue transplant or a celltransplant. In another embodiment, the cell transplant comprises bonemarrow cells or islet cells.

The invention provides the use of any of the disclosed CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules in the manufacture of a medicament fortreating any of the above stated diseases or disorders. The inventionalso provides the use of any of the disclosed CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules in a coadministration with another agentfor the treatment of the above-mentioned diseases or disorders. TheCTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules of the invention can beadministered to a subject, for example, intravenously, subcutaneously,and/or by inhalation. CTLA4-Ig or CTLA4^(A29YL104E)-Ig formulationsapplicable for intravenous or subcutaneous administration are describedin U.S. Ser. No. 60/752,150, filed on Dec. 20, 2005, which is herebyincorporated by reference in its entirety. CTLA4-Ig orCTLA4^(A29YL104E)-Ig formulations can also include liposome-basedformulations wherein the liposomes can deliver CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules to target cells or tissues. CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules can also be delivered to target cells ortissues by administration of a virus vector that comprises a CTLA4-Ig orCTLA4^(A29YL104E)-Ig gene expression cassette. Administration anddosages of a CTLA4-Ig or CTLA4^(A29YL104E)-Ig population of moleculesare described in U.S. Patent applications published as US20030083246 andUS20040022787, as well as the pending U.S. patent application serialnumber 60/668,774, filed on Apr. 6, 2005, all of which are herebyincorporated by reference in their entireties.

The CTLA4-Ig or CTLA4^(A29YL104E)-Ig molecules described herein may bein a variety of dosage forms which include, but are not limited to,liquid solutions or suspensions, tablets, pills, powders, suppositories,polymeric microcapsules or microvesicles, liposomes, and injectable orinfusible solutions. The form depends upon the mode of administrationand the therapeutic application. An effective mode of administration anddosage regimen for the molecules of the present invention depends uponthe severity and course of the disease, the subject's health andresponse to treatment and the judgment of the treating physician.Accordingly, the dosages of the molecules should be titrated to theindividual subject. The interrelationship of dosages for animals ofvarious sizes and species and humans based on mg/m² of surface area isdescribed by Freireich, E. J., et al. (Quantitative Comparison ofToxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey andMan. Cancer Chemother, Rep., 50, No.4, 219-244, May 1966). Adjustmentsin the dosage regimen may be made to optimize the growth inhibitingresponse.

Doses may be divided and administered on a daily basis or the dose maybe reduced proportionally depending upon the situation. For example,several divided doses may be administered daily or monthly or the dosemay be proportionally reduced as indicated by the specific therapeuticsituation. In one embodiment, the administration is monthly, quarterly,daily, twice a day, about every 10 hours, about every 6 hours, aboutevery 4 hours, about every 2 hours, about once an hour. In accordancewith the practice of the invention an effective amount for treating asubject may be between about 0.1 and about 10 mg/kg body weight ofsubject. Also, the effective amount may be an amount between about 1 andabout 10 mg/kg body weight of subject. The CTLA4-Ig orCTLA4^(A29YL104E)-Ig molecules of the invention also have in vivoclinical application. They can be used for the enumeration of B7positive cells in the diagnosis or prognosis of some conditions ofimmunodeficiency, the phenotyping of leukemias and lymphomas, and themonitoring of immunological change following organ transplantation.

The delivery of the compositions described herein may be achieved viainjection, oral delivery, inhalation of a spray or other particulardispersion, subcutaneous injection, intravenous delivery, topicaldelivery, suppository, ocular delivery, nasal or oral delivery. Thecomposition can be delivered via encapsulation in a liposome or othermembrane-like delivery vehicle. The composition can be delivered viablood or other fluids that are previously treated with the compositionand then subsequently transfused into a subject.

Sequence Listings

SEQ ID NO: 17 is the nucleotide sequence encoding the pcSDhuCTLA4Ig:GATCTCCCGA TCCCCTATGG TCGACTCTCA GTACAATCTG CTCTGATGCC GCATAGTTAAGCCAGTATCT GCTCCCTGCT TGTGTGTTGG AGGTCGCTGA GTAGTGCGCG AGCAAAATTTAAGCTACAAC AAGGCAAGGC TTGACCGACA ATTGCATGAA GAATCTGCTT AGGGTTAGGCGTTTTGCGCT GCTTCGCGAT GTACGGGCCA GATATACGCG TTGACATTGA TTATTGACTAGTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCGTTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGACGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAATGGGTGGACTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAAGTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACATGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCATGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGATTTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGGACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTACGGTGGGAGGT CTATATAAGC AGAGCTCTCT GGCTAACTAG AGAACCCACT GCTTACTGGCTTATCGAAAT TAATACGACT CACTATAGGG AGACCCAAGC TTGGTACCGA GCTCGGATCCACTAGTAACG GCCGCCAGTG TGCTGGAATT CTGCAGATAG CTTCACCAAT GGGTGTACTGCTCACACAGA GGACGCTGCT CAGTCTGGTC CTTGCACTCC TGTTTCCAAG CATGGCGAGCATGGCAATGC ACGTGGCCCA GCCTGCTGTG GTACTGGCCA GCAGCCGAGG CATCGCCAGCTTTGTGTGTG AGTATGCATC TCCAGGCAAA GCCACTGAGG TCCGGGTGAC AGTGCTTCGGCAGGCTGACA GCCAGGTGAC TGAAGTCTGT GCGGCAACCT ACATGATGGG GAATGAGTTGACCTTCCTAG ATGATTCCAT CTGCACGGGC ACCTCCAGTG GAAATCAAGT GAACCTCACTATCCAAGGAC TGAGGGCCAT GGACACGGGA CTCTACATCT GCAAGGTGGA GCTCATGTACCCACCGCCAT ACTACCTGGG CATAGGCAAC GGAACCCAGA TTTATGTAAT TGATCCAGAACCGTGCCCAG ATTCTGATCA GGAGCCCAAA TCTTCTGACA AAACTCACAC ATCCCCACCGTCCCCAGCAC CTGAACTCCT GGGGGGATCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAGGACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG TGGTGGTGGA CGTGAGCCACGAAGACCCTG AGGTCAAGTT CAACTGGTAC GTGGACGGCG TGGAGGTGCA TAATGCCAAGACAAAGCCGC GGGAGGAGCA GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTCCTGCACCAGG ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTCCCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTGTACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC AGGTCAGCCT GACCTGCCTGGTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG AGAGCAATGG GCAGCCGGAGAACAACTACA AGACCACGCC TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGCAAGCTCACCG TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATGCATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGTAAATGAGTGCGACGGC CGGCAAGCCC CCGCTCCCCG GGCTCTCGCG GTCGCACGAG GATGCTTCTAGAGGGCCCTA TTCTATAGTG TCACCTAAAT GCTAGAGCTC GCTGATCAGC CTCGACTGTGCCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAAGGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGTAGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAAGACAATAGCA GGCATGCTGG GGATGCGGTG GGCTCTATGG CTTCTGAGGC GGAAAGAACCAGCTGGGGCT CTAGGGGGTA TCCCCACGCG CCCTGTAGCG GCGCATTAAG CGCGGCGGGTGTGGTGGTTA CGCGCAGCGT GACCGCTACA CTTGCCAGCG CCCTAGCGCC CGCTCCTTTCGCTTTCTTCC CTTCCTTTCT CGCCACGTTC GCCCTGTGGA ATGTGTGTCA GTTAGGGTGTGGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCAGCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCATCTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCGCCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCCGAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTAGGCTTTTGCA AAAAGCTTGG ACAGCTGAGG GCTGCGATTT CGCGCCAAAC TTGACGGCAATCCTAGCGTG AAGGCTGGTA GGATTTTATC CCCGCTGCCA TCATGGTTCG ACCATTGAACTGCATCGTCG CCGTGTCCCA AGATATGGGG ATTGGCAAGA ACGGAGACCT ACCCTGGCCTCCGCTCAGGA ACGAGTTCAA GTACTTCCAA AGAATGACCA CAACCTCTTC AGTGGAAGGTAAACAGAATC TGGTGATTAT GGGTAGGAAA ACCTGGTTCT CCATTCCTGA GAAGAATCGACCTTTAAAGG ACAGAATTAA TATAGTTCTC AGTAGAGAAC TCAAAGAACC ACCACGAGGAGCTCATTTTC TTGCCAAAAG TTTGGATGAT GCCTTAAGAC TTATTGAACA ACCGGAATTGGCAAGTAAAG TAGACATGGT TTGGATAGTC GGAGGCAGTT CTGTTTACCA GGAAGCCATGAATCAACCAG GCCACCTCAG ACTCTTTGTG ACAAGGATCA TGCAGGAATT TGAAAGTGACACGTTTTTCC CAGAAATTGA TTTGGGGAAA TATAAACTTC TCCCAGAATA CCCAGGCGTCCTCTCTGAGG TCCAGGAGGA AAAAGGCATC AAGTATAAGT TTGAAGTCTA CGAGAAGAAAGACTAACAGG AAGATGCTTT CAAGTTCTCT GCTCCCCTCC TAAAGCTATG CATTTTTATAAGACCATGGG ACTTTTGCTG GCTTTAGATC TTTGTGAAGG AACCTTACTT CTGTGGTGTGACATAATTGG ACAAACTACC TACAGAGATT TAAAGCTCTA AGGTAAATAT AAAATTTTTAAGTGTATAAT GTGTTAAACT ACTGATTCTA ATTGTTTGTG TATTTTAGAT TCCAACCTATGGAACTGATG AATGGGAGCA GTGGTGGAAT GCCTTTAATG AGGAAAACCT GTTTTGCTCAGAAGAAATGC CATCTAGTGA TGATGAGGCT ACTGCTGACT CTCAACATTC TACTCCTCCAAAAAAGAAGA GAAAGGTAGA AGACCCCAAG GACTTTCCTT CAGAATTGCT AAGTTTTTTGAGTCATGCTG TGTTTAGTAA TAGAACTCTT GCTTGCTTTG CTATTTACAC CACAAAGGAAAAAGCTGCAC TGCTATACAA GAAAATTATG GAAAAATATT CTGTAACCTT TATAAGTAGGCATAACAGTT ATAATCATAA CATACTGTTT TTTCTTACTC CACACAGGCA TAGAGTGTCTGCTATTAATA ACTATGCTCA AAAATTGTGT ACCTTTAGCT TTTTAATTTG TAAAGGGGTTAATAAGGAAT ATTTGATGTA TAGTGCCTTG ACTAGAGATC ATAATCAGCC ATACCACATTTGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAAAATGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAGCAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTTGTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCGGC TGGATGATCC TCCAGCGCGGGGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT ATTGCAGCTT ATAATGGTTACAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAGTTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC TGTATACCGT CGACCTCTAGCTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCACAATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGTGAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTCGTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCGCTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGTATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAAGAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGCGTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAGGTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGTGCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGGAAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCGCTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGGTAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCACTGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTGGCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGTTACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGGTGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCCTTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTTGGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTTTAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAGTGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGTCGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACCGCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGCCGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCGGGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTACAGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACGTCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCCCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACTCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTCAACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAATACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTCTTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCACTCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAAAACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACTCATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGGATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCGAAAAGTGCCA CCTGACGTCG ACGGATCGGG A SEQ ID NO: 18 is the amino acidsequence of the extracellular domain of human CTLA4.MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD

Further Non-Limiting Embodiments

The invention provides for a clonal Chinese Hamster Ovary cellpopulation capable of producing CTLA4-Ig. In one embodiment, the cellpopulation is capable of producing greater than 0.5 or more grams ofCTLA4-Ig protein per liter of liquid culture, and wherein the CTLA4-Igexhibits a molar ratio of sialic acid to CTLA4-Ig dimer is from about 6to about 14 at a culture scale of 1,000 L or more. In one embodiment,the cell population has been adapted to serum-free, chemically definedmedium. In another embodiment, CTLA4-Ig produced from culture of thecell population has an extinction coefficient of 1.00±0.05 AU mL cm⁻¹mg⁻¹. In a further embodiment, the cell population, when grown inculture, is capable of producing CTLA4-Ig polypeptides, wherein: (a)about 90% of the CTLA4-Ig polypeptides comprise an amino acid sequenceof SEQ ID NO:2 beginning with the methionine at residue 27; (b) about10% of the CTLA4-Ig polypeptides comprise the amino acid sequence of SEQID NO:2 beginning with the alanine at residue number 26; (c) about 4% ofthe CTLA4-Ig polypeptides comprise the amino acid sequence of SEQ IDNO:2 ending with the lysine at residue number 383; (d) about 96% of theCTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2ending with the glycine at residue number 382; and optionally, (e) aboutless than 1% of the CTLA4-Ig polypeptides comprise the amino acidsequence of SEQ ID NO:2 beginning with the methionine at residue number25.

The invention provides for a progeny cell of the cells described above,wherein the progeny cell produces CTLA4-Ig. In one embodiment, theprogeny cell is obtained from culturing a cell over at least 5generations. In another embodiment, the progeny cell is obtained fromculturing a cell over at least 10 generations. In another embodiment,the progeny cell is obtained from culturing a cell over at least 20generations. In another embodiment,the progeny cell is obtained fromculturing a cell over at least 40 generations. In another embodiment,the progeny cell is obtained from culturing a cell over at least 50generations. In another embodiment, the progeny cell is obtained fromculturing a cell over at least 75 generations. In another embodiment,the progeny cell is obtained from culturing a cell over at least 100generations.

The invention provides for a cell line produced from any of the cellsdescribed above. In one embodiment, the cell line is clonal. In anotherembodiment, the cell line is capable of producing: (a) a CTLA4-Ig fusionprotein having an amino acid sequence of SEQ ID NO:8 (methionine atamino acid position 27 and glycine at amino acid position 382 of SEQ IDNO:2); (b) a CTLA4-Ig fusion protein having an amino acid sequence ofSEQ ID NO:5 (methionine at amino acid position 27 and lysine at aminoacid position 383 of SEQ ID NO:2); (c) a CTLA4-Ig fusion protein havingan amino acid sequence of SEQ ID NO:7 (alanine at amino acid position 26and glycine at amino acid position 382 of SEQ ID NO:2); (d) a CTLA4-Igfusion protein having an amino acid sequence of SEQ ID NO: 4 (alanine atamino acid position 26 and lysine at amino acid position 383 of SEQ IDNO:2); (e) a CTLA4-Ig fusion protein having an amino acid sequence ofSEQ ID NO:4 (methionine at amino acid position 25 and lysine at aminoacid position 383 of SEQ ID NO:2); or (f) a CTLA4-Ig fusion proteinhaving an amino acid sequence of SEQ ID NO:6 (methionine at amino acidposition 25 and glycine at amino acid position 382 of SEQ ID NO:2).

In another embodiment, the cell line is capable of producing CTLA4-Igfusion proteins, wherein: (a) about 90% of the CTLA4-Ig polypeptidescomprise an amino acid sequence of SEQ ID NO:2 beginning with themethionine at residue 27; (b) about 10% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 beginning with thealanine at residue number 26; (c) about 4% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 ending with the lysineat residue number 383; (d) about 96% of the CTLA4-Ig polypeptidescomprise the amino acid sequence of SEQ ID NO:2 ending with the glycineat residue number 382; and optionally, (e) about less than 1% of theCTLA4-Ig polypeptides comprise the amino acid sequence of SEQ ID NO:2beginning with the methionine at residue number 25.

In one embodiment, the CTLA4-Ig fusion proteins, which are produced fromculturing the cell line, have an extinction coefficient of 1.00±0.05 AUmL cm-1 mg-1. In one embodiment, the invention provides for a cellpopulation derived from a cell of the invention. In one embodiment, thecell population consists of at least one additional genetic change ascompared to the originally transfected cell and wherein the derived cellpopulation is capable of producing CTLA4-Ig. In other embodiments, thecell population consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 additionalgenetic changes as compared to the originally transfected cell andwherein the derived cell population is capable of producing CTLA4-Ig. Inone embodiment, the genetic change comprises at least onenon-conservative mutation in the cellular genome or in the recombinantexpression cassette encoding CTLA4-Ig.

In one embodiment, the genetic change comprises at least one additionalrecombinant nucleic acid within the cell. In one embodiment, the changecomprises a mutation of the cellular genome. In one embodiment, thechange comprises the addition of a nucleic acid to either the cellgenome or as a trans nucleic acid, which encodes an anti-apoptoticpolypeptide. In one embodiment, the anti-apoptotic polypeptide relatesto glycosylation.

In one embodiment, genetic change comprises at least one mutation of thecellular genome or of the recombinant expression cassette encodingCTLA4-Ig. In one embodiment, the cell population, when grown in culture,is capable of producing: (a) a CTLA4-Ig fusion protein having an aminoacid sequence of SEQ ID NO:8 (methionine at amino acid position 27 andglycine at amino acid position 382 of SEQ ID NO:2); (b) a CTLA4-Igfusion protein having an amino acid sequence of SEQ ID NO:5 (methionineat amino acid position 27 and lysine at amino acid position 383 of SEQID NO:2); (c) a CTLA4-Ig fusion protein having an amino acid sequence ofSEQ ID NO:7 (alanine at amino acid position 26 and glycine at amino acidposition 382 of SEQ ID NO:2); (d) a CTLA4-Ig fusion protein having anamino acid sequence of SEQ ID NO: 4 (alanine at amino acid position 26and lysine at amino acid position 383 of SEQ ID NO:2); (e) a CTLA4-Igfusion protein having an amino acid sequence of SEQ ID NO:4 (methionineat amino acid position 25 and lysine at amino acid position 383 of SEQID NO:2); or (f) a CTLA4-Ig fusion protein having an amino acid sequenceof SEQ ID NO:6 (methionine at amino acid position 25 and glycine atamino acid position 382 of SEQ ID NO:2).

The invention provides for a population of CTLA4-Ig molecules having anaverage molar ratio of sialic acid groups to CTLA4-Ig dimer of fromabout 6 to about 18. The invention provides for a population of CTLA4-Igmolecules having an average molar ratio of sialic acid groups toCTLA4-Ig dimer of from about 8 to about 18. The invention provides for apopulation of CTLA4-Ig molecules having an average molar ratio of sialicacid groups to CTLA4-Ig dimer of from about 11 to about 18. Theinvention provides for a population of CTLA4-Ig molecules having anaverage molar ratio of sialic acid groups to CTLA4-Ig dimer of fromabout 12 to about 18. The invention provides for a population ofCTLA4-Ig molecules having an average molar ratio of sialic acid groupsto CTLA4-Ig dimer of from about 13 to about 18. The invention providesfor a population of CTLA4-Ig molecules having an average molar ratio ofsialic acid groups to CTLA4-Ig dimer of from about 14 to about 18. Theinvention provides for a population of CTLA4-Ig molecules having anaverage molar ratio of sialic acid groups to CTLA4-Ig dimer of fromabout 15 to about 17. The invention provides for a population ofCTLA4-Ig molecules having an average molar ratio of sialic acid groupsto CTLA4-Ig dimer of about 16.

The invention provides for a population of CTLA4-Ig molecules, whereingreater than 95% of the molecules are CTLA4-Ig dimers. In oneembodiment, greater than 98% of the molecules are CTLA4-Ig dimers. Inone embodiment, greater than 99% of the molecules are CTLA4-Ig dimers.In one embodiment, greater than 99.5% of the molecules are CTLA4-Igdimers. In one embodiment, from about 95% to about 99.5% of themolecules are CTLA4-Ig dimers and about 0.5% to about 5% of themolecules are CTLA4-Ig tetramers. In one embodiment, about 98.6% of themolecules are CTLA4-Ig dimers and about 1.2% of the molecules areCTLA4-Ig tetramers and about less than 0.7% of the molecules areCTLA4-Ig monomers. The invention provides for a population consisting ofCTLA4-Ig dimers. The invention provides for a population of CTLA4-Igmolecules, wherein the population is substantially free of CTLA4-Igmonomer. The invention provides for a population of CTLA4-Ig molecules,wherein the population is substantially free of CTLA4-Ig tetramer. Theinvention provides for a population of CTLA4-Ig monomer moleculessubstantially free of CTLA4-Ig dimer and tetramer. In one embodiment,each monomer of each CTLA4-Ig dimer has at least 3 sialic acid groups.

In one embodiment, each monomer of eachCTLA4-Ig dimer has from at least3 sialic acid groups to at least 8 sialic acid groups. The inventionprovides for a purified population of CTLA4-Ig tetramer molecules, thepopulation being substantially free of CTLA4-Ig dimer, and optionallywherein the population comprises an amount that is greater than about100 grams. The invention provides for a purified population of CTLA4-Igtetramer molecules, the population being substantially free of CTLA4-Igmonomer, and optionally wherein the population comprises an amount thatis greater than about 100 grams. In one embodiment, each tetramermolecule comprises two pairs of CTLA4-Ig polypeptides, wherein eachpolypeptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 3-8, and wherein each member of the pair ofpolypeptides is covalently linked to the other member, and wherein thetwo pairs of polypeptides are non-covalently associated with oneanother. In one embodiment, each tetramer molecule is capable of bindingto a CD80 or CD86. In one embodiment, each tetramer molecule has atleast a 2-fold greater avidity for CD80 or CD86 as compared to aCTLA4-Ig dimer molecule. In one embodiment, each tetramer molecule hasat least a 2-fold greater inhibition of T cell proliferation oractivation as compared to a CTLA4-Ig dimer molecule.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein the composition comprises dominant isoforms visualizable on anisoelectric focusing gel of CTLA4-Ig which have an isoelectric point,pI, less than or equal to 5.1 as determined by isoelectric focusing. Inone embodiment, the pI increases after neuraminidase treatment. In oneembodiment, at least 40% of the CTLA4-Ig molecules exhibit anisoelectric point less than or equal to about 5.1 as determined byisoelectric focusing. In one embodiment, at least 70% of the CTLA4-Igmolecules exhibit an isoelectric point less than or equal to about 5.1as determined by isoelectric focusing. In one embodiment, at least 90%of the CTLA4-Ig molecules exhibit an isoelectric point less than orequal to about 2.5 as determined by isoelectric focusing. The inventionprovides for a population of CTLA4-Ig molecules having a pI of fromabout 2.0±0.2 to about 5.0±0.2. The invention provides for a populationof CTLA4-Ig molecules having a pI from about 4.3±0.2 to about 5.0±0.2.The invention provides for a population of CTLA4-Ig molecules having apI of about 3.3±0.2 to about 4.7±0.2. The invention provides for amethod for preparing a composition, the composition comprising aCTLA4-Ig molecule with a pI of from about 2.0±0.2 to about 5.0±0.2, themethod comprising: (a) subjecting a mixture of CTLA4-Ig molecules toisoelectric focusing gel electrophoresis, wherein a single band on thegel represents a population of CTLA4-Ig molecules with a particular pI,and (b) isolating the population of CTLA4-Ig molecules having a pI offrom about 2.0±0.2 to about 5.0±0.2 so as to prepare the composition.The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of GlcNAc per mole of CTLA4-Ig dimer of from about 17 to about 25.The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of GlcNAc per mole of CTLA4-Ig dimer of from about 15 to about 35.The invention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA4-Ig molecules are characterized by an average molarratio of GalNAc per mole of CTLA4-Ig dimer of from about 1.7 to about3.6. The invention provides for a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules are characterized by anaverage molar ratio of galcatose per mole of CTLA4-Ig dimer of fromabout 8 to about 17. The invention provides for a composition comprisingCTLA4-Ig molecules, wherein the CTLA4-Ig molecules are characterized byan average molar ratio of fucose per mole of CTLA4-Ig dimer of fromabout 3.5 to about 8.3. The invention provides for a compositioncomprising CTLA4-Ig molecules, wherein the CTLA4-Ig molecules arecharacterized by an average molar ratio of mannose per mole of CTLA4-Igdimer of from about 7.2 to about 22. The invention provides for acomposition comprising CTLA4-Ig molecules, wherein the CTLA4-Igmolecules are characterized by an average molar ratio of sialic acid permole of CTLA4-Ig dimer of from about 6 to about 12. The inventionprovides for a composition comprising CTLA4-Ig molecules characterizedby: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer fromabout 15 to about 35; and (b) an average molar ratio of sialic acid permole of CTLA4-Ig dimer from about 6 to about 12. The invention providesfor a composition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Igdimer from about 1.7 to about 3.6; and (c) an average molar ratio ofsialic acid per mole of CTLA4-Ig dimer from about 6 to about 12. Theinvention provides for a composition comprising CTLA4-Ig moleculescharacterized by: (a) an average molar ratio of GlcNAc per mole ofCTLA4-Ig dimer from about 15 to about 35; (b) an average molar ratio ofGalNAc per mole CTLA4-Ig dimer from about 1.7 to about 3.6; (c) anaverage molar ratio of galcatose per mole CTLA4-Ig dimer from about 8 toabout 17; and (d) an average molar ratio of sialic acid per mole ofCTLA4-Ig dimer from about 6 to about 12. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Igdimer from about 1.7 to about 3.6; (c) an average molar ratio ofgalcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) anaverage molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 toabout 8.3; and (e) an average molar ratio of sialic acid per mole ofCTLA4-Ig dimer from about 6 to about 12. The invention provides for acomposition comprising CTLA4-Ig molecules characterized by: (a) anaverage molar ratio of GlcNAc per mole of CTLA4-Ig dimer from about 15to about 35; (b) an average molar ratio of GalNAc per mole CTLA4-Igdimer from about 1.7 to about 3.6; (c) an average molar ratio ofgalcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) anaverage molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 toabout 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimerfrom about 7.2 to about 22; and (f) an average molar ratio of sialicacid per mole of CTLA4-Ig dimer from about 6 to about 12.

The invention provides for a composition comprising CTLA4-Ig molecules,wherein composition exhibits an NGNA chromatogram peak of about9.589+/−0.3 and an NANA chromatogram peak of about 10.543+/-0.3. Theinvention provides for a composition comprising CTLA4-Ig molecules,wherein the CTLA-Ig molecules exhibit a carbohydrate profile as shown inFIG. 7. The invention provides for a composition comprising CTLA4-Igmolecules, wherein the CTLA4-Ig molecules exhibit a carbohydrate profileof Domains I-IV, wherein Domain I comprises peaks which representa-sialylated oligosaccharides, Domain II comprises peaks which representmono-sialylated oligosaccharides, Domain III comprises peaks whichrepresent di-sialylated oligosaccharides, and Domain IV comprises peakswhich represent tri-sialylated oligosaccharides. In one embodiment, thedifference in retention times of N-linked oligosaccharides between afirst peak in Domain I and a main peak in Domain II is from about 22 toabout 28 minutes. The invention provides for a composition comprisingCTLA4-Ig dimer molecules, wherein at least 0.5% of the CTLA4-Ig dimermolecules are cysteinylated. In another embodiment, at least 1.0% of theCTLA4-Ig dimer molecules are cysteinylated. The invention provides for apopulation of CTLA4-Ig molecules, wherein the population exhibits a massspectrometry profile as shown in FIG. 8. The invention provides for apopulation of CTLA4-Ig molecules, wherein the population exhibits acapillary electrophoresis profile as shown in FIGS. 19 and 20. Theinvention provides for a composition of CTLA4-Ig molecules having anaverage molar ratio of sialic acid groups to CTLA4-Ig dimer of fromabout 6 to about 18, wherein the CTLA4-Ig dimer is produced from cellsof a commercial cell line. The invention provides for a CTLA4-Igcomposition obtained by any method of the invention. The inventionprovides for a population of CTLA4-Ig molecules, wherein the moleculesare glycosylated at an aparagine amino acid residue at position 102 ofSEQ ID NO:2, an aparagine amino acid residue at position 134 of SEQ IDNO:2, an aparagine amino acid residue at position 233 of SEQ ID NO:2, aserine amino acid residue at position 155 of SEQ ID NO:2, or a serineamino acid residue at position 165 of SEQ ID NO:2. The inventionprovides for a population of CTLA4-Ig molecules, wherein the populationof molecules is characterized by: (a) an average molar ratio of GlcNAcper mole of CTLA4-Ig dimer from about 15 to about 35; (b) an averagemolar ratio of GalNAc per mole CTLA4-Ig dimer from about 1.7 to about3.6; (c) an average molar ratio of galcatose per mole CTLA4-Ig dimerfrom about 8 to about 17; (d) an average molar ratio of fucose per moleCTLA4-Ig dimer from about 3.5 to about 8.3; (e) an average molar ratioof mannose per mole CTLA4-Ig dimer from about 7.2 to about 22;(f) anaverage molar ratio of sialic acid per mole of CTLA4-Ig dimer from about6 to about 12; (g) a pI as determined from visualization on anisoelectric focusing gel in a range from about 2.4±0.2 to about 5.0±0.2;(h) MCP-1 of less than or equal to 5 ppm;(i) less than 2.5% tetramer;(j) less than 0.5% monomer; (k) CTLA4-Ig polypeptides of the populationhaving an amino acid at least 95% identical to any of SEQ ID NOS:2-8;(1) wherein CTLA4-Ig molecules within the population is capable ofbinding to CD80 and CD86. The invention provides for a population ofCTLA4-Ig molecules, wherein the population of molecules is characterizedby: (a) an average molar ratio of GlcNAc per mole of CTLA4-Ig dimer fromabout 15 to about 35; (b) an average molar ratio of GalNAc per moleCTLA4-Ig dimer from about 1.7 to about 3.6; (c) an average molar ratioof galcatose per mole CTLA4-Ig dimer from about 8 to about 17; (d) anaverage molar ratio of fucose per mole CTLA4-Ig dimer from about 3.5 toabout 8.3; (e) an average molar ratio of mannose per mole CTLA4-Ig dimerfrom about 7.2 to about 22; (f) an average molar ratio of sialic acidper mole of CTLA4-Ig dimer from about 6 to about 12; (g) a pI asdetermined from visualization on an isoelectric focusing gel in a rangefrom about 2.4±0.2 to about 5.0±0.2; (h) MCP-1 of less than or equal to5 ppm; (i) less than 2.5% tetramer; (j) less than 0.5% monomer; (k)CTLA4-Ig polypeptides of the population having an amino acid at least95% identical to any of SEQ ID NOS: 2-8; (1) wherein CTLA4-Ig moleculeswithin the population is capable of binding to CD80 and CD86; orpharmaceutical equivalents thereof The invention provides for acomposition comprising an effective amount of the CTLA4-Ig molecules anda pharmaceutically acceptable carrier. The invention provides for acomposition comprising an effective amount of the CTLA4-Ig molecules,wherein the composition further comprises an amount of maltosemonohydrate. In one embodiment, the composition further comprises apharmaceutically acceptable diluent, adjuvant or carrier. In oneembodiment, the composition further comprises maltose, sodium phosphatemonobasic monohydrate, sodium chloride, sodium hydroxide, and sterilewater. In one embodiment, the composition further comprises sucrose,poloxamer, sodium phosphate monobasic monohydrate, sodium phosphatedibasic anhydrous, sodium chloride, sodium hydroxide, and sterile water.

The invention provides for a lyophilized CTLA4-Ig mixture comprising atleast 95% CTLA4-Ig dimer, and not more than 5% CTLA4-Ig tetramer. In oneembodiment, the mixture comprises at least 98% CTLA4-Ig dimer and nomore than 2% CTLA4-Ig tetramer. In one embodiment, the mixture comprisesat least 99% CTLA4-Ig dimer and no more than 1% CTLA4-Ig tetramer. Inone embodiment, the mixture comprises at least 8.0 moles of sialic acidper mole of CTLA4-Ig dimer. In one embodiment, the mixture comprisesfrom about 15.7 to about 31 moles of GlcNAc per mole of CTLA4-Ig dimer.In one embodiment, the mixture comprises from about 1.6 to about 3.2moles of GalNAc per mole of CTLA4-Ig dimer. In one embodiment, themixture comprises from about 9.3 to about 15.5 moles of galactose permole of CTLA4-Ig dimer. In one embodiment, the mixture comprises fromabout 3.6 to about 7.9 moles of fucose per mole of CTLA4-Ig dimer. Inone embodiment, the mixture comprises from about 9.7 moles of mannoseper mole of CTLA4-Ig dimer. The invention provides for a pharmaceuticalkit comprising: (a) a container containing a lyophilized CTLA4-Igmixture of claim 1; and (b) instructions for reconstituting thelyophilized CTLA4-Ig mixture into solution for injection.

The invention provides for a method for inhibiting T cell proliferation(or activation), the method comprising contacting a T cell with aneffective amount of the CTLA4-Ig composition. The invention provides fora method for inhibiting an immune response in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of the composition. The invention provides methods for inducingimmune tolerance to an antigen in a subject, treating inflammation in asubject, treating rheumatoid arthritis, treating psoriasis in a subject,treating lupus in a subject, treating or preventing an allergy in asubject, treating or preventing graft vs host disease in a subject,treating or preventing rejection of a transplanted organ in a subject,treating multiple sclerosis in a subject, treating type I diabetes in asubject, treating inflammatory bowel disease in a subject, treatingoophoritis in a subject, treating glomerulonephritis in a subject,treating allergic encephalomyelitis in a subject, or treating myastheniagravis in a subject by administering a composition of the invention inan amount to a subject to treat the disease or disorder. The compositionmay be combined with a pharmaceutically acceptable carrier. Theinvention provides for the use of a population of CTLA4-Ig moleculeshaving an average molar ratio of sialic acid groups to CTLA4-Ig dimer offrom about 6 to about 18 in the manufacture of a medicament for thetherapeutic and/or prophylactic treatment of an immune disorder. Theinvention provides for the use of a population of CTLA4-Ig moleculeshaving an average molar ratio of sialic acid groups to CTLA4-Ig dimer offrom about 6 to about 18 in the manufacture of an anti-rheumatoidarthritis agent in a package together with instructions for its use inthe treatment of rheumatoid arthritis. The invention provides for amethod for inhibiting T cell proliferation (or activation), the methodcomprising contacting a T cell with an effective amount of thecomposition of the invention in combination with methotrexate. Theinvention provides for a method for inhibiting an immune response in asubject, the method comprising administering to a subject in needthereof an effective amount of the composition of the invention incombination with methotrexate. The invention provides for a method forinducing immune tolerance to an antigen in a subject, the methodcomprising administering to a subject in need thereof an effectiveamount of the composition of any of claims 1-64 in combination withmethotrexate. The invention provides for a method for producing arecombinant protein, the method comprising: (a) expanding mammaliancells that secrete a recombinant protein, wherein the expanding is froma seed culture to a liquid culture of at least 10,000 L, wherein therecombinant protein concentration is at least 0.5 grams/L of liquidculture; and (b) isolating the recombinant protein from the at least10,000 L liquid culture. In one embodiment, the expanding of step (a)comprises: (i) culturing the cells in a serum-free, chemically definedmedium with at least four passages so as to obtain a cell density of atleast about 1.0×10⁵ viable cells per mL, wherein each seed stage startsat about 2×10⁵ per ml and goes to 1-2 mil cells per ml; (ii) maintainingthe cells in culture for a time sufficient to produce from the cultureat least about 0.5 g/L. In one embodiment, the protein is aglycoprotein. In one embodiment, the protein is a CTLA4-Ig protein. Inone embodiment, the mammalian cells are progeny cells. In oneembodiment, the mammalian cells are progeny of a CHO clonal cell linecapable of producing CTLA4-Ig fusion protein, wherein the CHO cells havestably integrated in their genome at least 30 copies of a CTLA4-Igexpression cassette. In one embodiment, the time sufficient is a time bywhich the cells' viability does not fall below 30%. In one embodiment,the time sufficient is a time by which the cells' viability does notfall below 40%. In one embodiment, the time sufficient is a time bywhich the cells' viability does not fall below 50%. In one embodiment,the time sufficient is a time by which the cells' viability does notfall below 60%. In one embodiment, the time sufficient is a time bywhich the cells' viability does not fall below 70%, or 80% or 90% or95%. In one embodiment, the at least four passages comprises: (i)growing the cells in a culture volume of at least 50 mL until a celldensity of from about 1 million to about 2.5 mill cells per ml isreached, (ii) growing the cells in a culture volume of at least 10 Luntil a cell density of about 1 million to about 2.5 million cells perml is reached; (iii) growing the cells in a culture volume of at least100 L until a cell density of about 1 million to about 2.5 million cellsper ml is reached; and (iv) growing the cells in a culture volume of 200L until a cell density of about 1 million to about 2.5 million cells perml is reached. In one embodiment, galactose is added to the serum-free,chemically defined medium. In one embodiment, the maintaining comprises(i) lowering the temperature of the culture from 37±2° C. to 34±2° C.;and (ii) lowering the temperature of the culture from 34±2° C. to 32±2°C. In one embodiment, the temperature is kept within the range of 32±2°C. for at least 5 days. In one embodiment, the temperature is keptwithin the range of 32±2° C. for at least 6 days. In one embodiment, thetemperature is kept within the range of 32±2° C. for at least 7 days. Inone embodiment, the temperature is kept within the range of 32±2° C. forat least 8 days. In one embodiment, the temperature is kept within therange of 32±2° C. for at least 9 days. In one embodiment, thetemperature is kept within the range of 32±2° C. for at least 10 days.In one embodiment, the temperature is kept within the range of 32±2° C.for at least 11 days. In one embodiment, the temperature is kept withinthe range of 32±2° C. for at least 12 days. In one embodiment, thetemperature is kept within the range of 32±2° C. for at least 13 days.In one embodiment, the temperature is kept within the range of 32±2° C.for at least 14 days. In one embodiment, the temperature is kept withinthe range of 32±2° C. for at least 15 days. In one embodiment, thetemperature is kept within the range of 32±2° C. for at least 16 days.In one embodiment, the temperature is kept within the range of 32±2° C.for at least 17 days. In one embodiment, the temperature is kept withinthe range of 32±2° C. for at least 18 days. In one embodiment, thetemperature is kept within the range of 32±2° C. for up to 18 days. Inone embodiment, the temperature is kept within the range of 32±2° C.until the cell density of the culture is from about 30×10⁵ to about79×10⁵ cells per mL of liquid culture. The invention provides for amethod for producing a recombinant protein, the method comprising: (a)expanding mammalian cells that secrete a recombinant protein from a seedculture to a liquid culture of at least 10,000 L so that the recombinantprotein concentration is at least 0.5 grams/L of liquid culture; and (b)isolating the recombinant protein from the at least 10,000 L liquidculture, wherein the isolating occurs only when the liquid culturecontains greater than or equal to about 6.0 moles of NANA per mole ofprotein. The invention provides for a method for producing a recombinantprotein, the method comprising: (a) expanding mammalian cells thatsecrete a recombinant protein from a seed culture to a liquid culture ofat least 10,000 L so that the recombinant protein concentration is atleast 0.5 grams/L of liquid culture; and (b) isolating the recombinantprotein from the at least 10,000 L liquid culture, wherein the isolatingoccurs only when the liquid culture has a cell density of from about33×10⁵ to about 79×10⁵ cells per mL.

The invention provides for a method for producing a recombinant protein,the method comprising: (a) expanding mammalian cells that secrete arecombinant protein from a seed culture to a liquid culture of at least10,000 L so that the recombinant protein concentration is at least 0.5grams/L of liquid culture; and (b) isolating the recombinant proteinfrom the at least 10,000 L liquid culture, wherein the isolating occurswhen cell viability in the liquid culture has not fallen below about20%, or about 30%, or about 38%. The invention provides for a method forproducing a recombinant protein, the method comprising: (a) expandingmammalian cells that secrete a recombinant protein from a seed cultureto a liquid culture of at least 10,000 L so that the recombinant proteinconcentration is at least 0.5 grams/L of liquid culture; and (b)isolating the recombinant protein from the at least 10,000 L liquidculture, wherein the isolating occurs only when endotoxin is less thanor equal to about 76.8 EU per mL of liquid culture. The inventionprovides for a method for producing a recombinant protein, the methodcomprising: (a) expanding mammalian cells that secrete a recombinantprotein from a seed culture to a liquid culture of at least 10,000 L sothat the recombinant protein concentration is at least 0.5 grams/L ofliquid culture; and (b) isolating the recombinant protein from the atleast 10,000 L liquid culture, wherein the isolating occurs only whenbioburden is less than 1 colony forming unit per mL of liquid culture.The invention provides for a method for producing a recombinant protein,the method comprising: (a) expanding mammalian cells that secrete arecombinant protein from a seed culture to a liquid culture of at least10,000 L so that the recombinant protein concentration is at least 0.5grams/L of liquid culture; and (b) isolating the recombinant proteinfrom the at least 10,000 L liquid culture, wherein the isolating occursonly if at least two of the following conditions are met: (i) the liquidculture contains greater than or equal to about 6.0 moles of NANA permole of protein, (ii) the liquid culture has a cell density of fromabout 33×10⁵ to about 79×10⁵ cells per mL, (iii) cell viability in theliquid culture has not fallen below about 20%, or about 38%, or (iv)amount of CTLA4-Ig in the culture is greater than 0.5 g/L. In oneembodiment, the isolating comprises: (i) obtaining a cell culturesupernatent; (ii) subjecting the supernatant to anion exchangechromotagraphy to obtain an eluted protein product; (iii) subjecting theeluted protein product of step (ii) to hydrophobic interactionchromatography so as to obtain an enriched protein product; (iv)subjecting the enriched protein product to affinity chromatography toobtain an eluted and enriched protein product; and (v) subjecting theeluted and enriched protein product of (iv) to anion exchangechromatography. In one embodiment, the enriched protein product obtainedin step (iii) is characterized in that a percentage of any highmolecular weight multimer is less than 25% by weight. In one embodiment,the anion exchange chromatography of step (ii) is carried out using awash buffer comprising about 75 mM HEPES, and about 360 mM NaCl, andhaving a pH of about 8.0. In one embodiment, the anion exchangechromatography of step (ii) is carried out using an elution buffercomprising about 25 mM HEPES, and about 325 mM NaCl, and having a pH ofabout 7.0. In one embodiment, the hydrophobic interaction chromatographyof step (iii) is carried out using a single wash buffer comprising about25 mM HEPES, and about 850 mM NaCl, and having a pH of about 7.0. In oneembodiment, the affinity chromatography of step (iv) is carried outusing a wash buffer comprising about 25 mM Tris, and about 250 mM NaCl,and having a pH of about 8.0. In one embodiment, the affinitychromatography of step (iv) is carried out using an elution buffercomprising about 100 mM Glycine and having a pH of about 3.5. In oneembodiment, the anion exchange chromatography of step (v) is carried outusing a wash buffer comprising about 25 mM HEPES, and from about 120 mMNaCl to about 130 mM NaCl, and having a pH of about 8.0. In oneembodiment, the anion exchange chromatography of step (v) is carried outusing an elution buffer comprising about 25 mM HEPES, and about 200 mMNaCl, and having a pH of about 8.0. In one embodiment, the anionexchange chromatography of step (ii) is carried out using a columnhaving an anion exchange resin having a primary, secondary, tertiary, orquartenary amine functional group. In one embodiment, the resin has aquartenary amine functional group. In one embodiment, the hydrophobicinteraction chromatography of step (iii) is carried out using ahydrophobic interaction resin having a phenyl, an octyl, a propyl, analkoxy, a butyl, or an isoamyl functional group. In one embodiment, thefunctional group is a phenyl functional group. In one embodiment, theaffinity chromatography of step (iv) is carried out using a columncontaining Protein A. In one embodiment, method for preparing CTLA4-Ig,the method comprising purifying CTLA4-Ig from a liquid cell culture sothat the purified CTLA4-Ig (a) has about 38 ng of MCP-1 per mg ofCTLA4-Ig dimer, and (b) comprises less than 2.5% of CTLA4-Ig tetramer byweight. In one embodiment, the liquid cell culture comprises a cell ofor a progeny cell of the invention. The invention provides a method forproducing CTLA4-Ig, the method comprising: (a) expanding progeny cellsof any of commercial cell line or CHO cells that are capable ofproducing CTLA4-Ig, wherein the expanding is from a seed culture to aliquid culture of at least 10,000 L, wherein the CTLA4-Ig concentrationis at least 0.5 grams/L of liquid culture; and (b) isolating CTLA4-Igfrom the at least 10,000 L liquid culture, wherein the chromotagraphy ison a column with hydrophobic interaction resin with at least a phenylfunctional group, wherein the isolating comprises a step of hydrophobicinteraction chromatography carried out using a single wash buffercomprising about 25 mM HEPES, and about 850 mM NaCl, and having a pH ofabout 7.0.

EXAMPLES OF THE INVENTION

A number of Examples are provided below to facilitate a more completeunderstanding of the present invention. The following examplesillustrate the exemplary modes of making and practicing the presentinvention. However, the scope of the invention is not limited tospecific embodiments disclosed in these Examples, which are for purposesof illustration only, since alternative methods may be utilized toobtain similar results.

The following Examples refer to CTLA4-Ig molecules that comprisesequences of one or more of SEQ ID NOS: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15 or 16. These Examples are not meant to be limiting, and oneskilled in the art understands that the Examples can be expanded andadapted to, for example, other CTLA4-Ig molecules, other glycoproteins,and other proteins related to or comprising portions of anIg-superfamily protein.

The following table sets out examples that relate to CTLA4-Ig and toCTLA4^(A29YL104E)-Ig.

Exemplary Exemplary CTLA4- CTLA4-Ig Protein Ig Protein No. 2 -- No. 1 --CTLA4-Ig CTLA4^(A29YL104E)-Ig CTLA4-Ig Protein having SEQ ID NO: 1,having SEQ ID NO: Characteristics 2, 5, 6, 7, 8, 9, or 10 3 or 4 or11-16 Binding to B7-1; on/ Example 6 off rates; potency, valencyBioburden Example 49 Capillary Example 38 Electrophoresis CarbohydrateExample 3 Example 22 content, N-linked Example 44 HPEAC profile, Example37 Domains Cell line transfection Example 12 Example 23 CHO DNA Example58 Example 55 CHO Host cell Example 60 Example 52 protein GeneticExample 24 Characterization Disaggregation Example 5 Example 33Endotoxin Example 48 Example 48 Final fill Example 30 FormulationsExample 2 Example 27 GalNAc, GlcNAc Example 17, Example 63 Example 36molar ratios IEF Example 50 Example 22 IL-2 bioassay Example 45 Example40 Immunogenicity Example 31 Single dose healthy Example 66 PK MALDI-TOFExample 8 Mannose, fucose, Example 18 Example 35 galactose molar Example64 ratios Mass Spec Example 7 MCP-1 Example 59 Example 54Media/culturing Example 9 Monkey PK Example 32 Monomer Example 4 NANA,NGNA Example 3, Example 39 molar ratios Example 16 O-linked Example 46Oxidation and Example 47 Deamidation PK Correlations Example 42 PlasmidExample 1, Example 11, Example 67 Production Example 14 Example 19Example 28 Protein A Example 62 Example 53 Purification Example 15Example 20 Example 29 Multiple Dose RA Example 34 PK SDS-PAGE Example 51Example 26 Example 56 SEC - HMW, dimer, Example 10 Example 25 monomer,size homogeneity SPR - binding to Example 21 Example 41 B7.1 (BIAcore)Sub-cloning of cell Example 13 Example 24 lines Triton-X 100 Example 61Example 57 Trypic mappings Example 3, Example 47, Example 22 Example 65

Abbreviations:

-   15 N 15 Nanometer-   A₂₈₀ Absorbance at 280 nm-   CTLA4-Ig Cytotoxic T-Lymphocyte Antigen-4 Immunoglobulin; CTLA-4 Ig-   API Active Pharmaceutical Ingredient-   AU Absorbance Units-   B7 CTLA-4 Receptor Ligand-   cfu Colony Forming Unit-   CHO Chinese Hamster Ovary-   CHOP Chinese Hamster Ovary Host Cell Proteins-   CV Column Volume-   Drug Substance Fill Drug Substance Concentration/Diafiltration and    Fill Step-   ELISA Enzyme Linked Immunosorbent Assay-   EU Endotoxin Units-   Fc The constant region of antibodies-   GalNAc N-Acetyl-galactosamine-   GlcNAc N-Acetyl-glucosamine-   HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic Acid-   HIC Hydrophobic Interaction Chromatography; Phenyl Sepharose™ Fast    Flow-   HMW High Molecular Weight-   HPLC High Performance Liquid Chromatography-   IgG1 Immunoglobulin in Class G1-   IPC In-Process Control-   LAL Limulus Amebocyte Lysate-   MBR(s) Master Batch Record(s)-   MCP-1 Monocyte Chemotactic Protein 1-   MTX Methotrexate-   MW Molecular Weight-   N/A Not Applicable-   NANA N-acetylneuraminic Acid; Sialic Acid; SA-   NMWC Nominal Molecular Weight Cutoff-   OD Optical Density-   PAR Proven Acceptable Range-   Planova Viral removal filters, pore size 15 nm-   PP(s) Process Parameter(s)-   PQ Performance Qualification-   psi Pounds per Square Inch-   psid Pounds per Square Inch, Differential-   psig Pounds per Square Inch; Gauge-   QFF Q Sepharose™ Fast Flow-   QXL Q Sepharose Extreme Load-   rPA Recombinant Protein A Sepharose Fast Flow-   SA Sialic Acid; N-acetylneuraminic Acid; NANA-   SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis-   SOP Standard Operating Procedure-   Tris Tris (Hydroxymethyl) Aminomethane Triton X-100    t-Octylphenoxypolyethoxyethanol; Polyethylene glycol    tert-octylphenyl ether-   UF Ultrafiltration-   UV Ultraviolet-   v/v Volume per Volume-   VF Viral Filtration; Nanometer Filtration-   VI Viral Inactivation

Example 1 Confirmation of the CTLA4-Ig Coding Sequence in PlasmidpcSDhuCTLA4-Ig

The annotated nucleic acid sequence of the CTLA4-Ig gene present inpcSDhuCTLA4-Ig and the corresponding deduced amino acid sequence ofCTLA4-Ig are shown in FIG. 1. The pcSDhuCTLA4-Ig nucleic acid wastransfected into CHO cells in order to generate stable transfectantsthat could express CTLA4-Ig molecules (see Example 12). Thetransfectants were screened and certain transfectants were subcloned orexpanded to generate clonal cell lines.

Analysis of the DNA sequence data confirmed that the junctions createdduring the plasmid construction were as designed and that the syntheticoligonucleotide primers used in the polymerase chain reaction generatedthe correct oncostatin M signal sequence upstream of the CTLA4-Igsequence. The desired cysteine to serine changes (at positions 156, 162and 165 of SEQ ID NO:2) in the hinge region of the fusion protein wereconfirmed. These amino acid residues are designated in bold with anasterisk in FIG. 1. An additional amino acid change of proline to serineat position 174 of SEQ ID NO:2 was also detected. This change wasintroduced during the IgG₁ cDNA synthesis by the polymerase chainreaction. This amino acid residue is also designated in bold with anasterisk in FIG. 1.

The analysis of the DNA sequence data identified one additionaldifference when compared to the published nucleotide sequences of thehuman CTLA4 and human IgG₁ constant region. The codon at amino acid 110of the CTLA4 coding region was identified as ACC (threonine) rather thanGCC (alanine).

Example 2 CTLA4-Ig Formulation

CTLA4-Ig composition for Injection, 250 mg/vial, is a sterile,non-pyrogenic lyophile for intravenous (IV) administration. Thepopulation of CTLA4-Ig molecules is packaged in 15-cc Type I flinttubing glass vials. Each vial is stoppered with a 20-mm Daikyo graybutyl D-21-7-S/B2-TR fluoro-resin coated stopper and sealed with a 20-mmaluminum, white, flip-off seal.

Each single-use vial contains 250 mg of CTLA4-Ig composition which isconstituted with Sterile Water for Injection, USP and further dilutedwith 0.9% Sodium Chloride Injection, USP, at the time of use. Thecomposition of CTLA4-Ig for Injection, 250 mg/vial, and the function ofeach component is listed in the Table below.

TABLE 8 Composition of CTLA4-Ig Component Function mg per Vial CTLA4-IgActive Ingredient 262.5 Maltose Monohydrate Bulking Agent/ 525Stabilizer Sodium Phosphate, Monobasic, Buffering Agent 18.1Monohydrate^(b) Sodium Chloride^(b) Ionic Strength 15.3 AdjustmentHydrochloric Acid pH Adjustment adjust pH to ~7.5 Sodium Hydroxide pHAdjustment ^(b)These components are present in the CTLA4-Ig compositionsolution

CTLA4-Ig composition contains approximately 50 mg/mL CTLA4-Ig in 25 mMsodium phosphate buffer and 50 mM sodium chloride at pH 7.5. Duringearly development, this buffer system was selected based on theevaluation of the physical and chemical stability of CTLA4-Ig as afunction of pH, buffer type, buffer concentration and sodium chlorideconcentration. The stability of CTLA4-Ig solutions was investigated inthe pH range of 5 to 9. The results indicated that the formation of highmolecular weight species was pH dependent and the pH range of maximumstability was between 7 and 8. In a separate determination, the effectof buffer type and concentration was evaluated, where the CTLA4-Igcomposition was found to be equally stable in sodium phosphate or trisbuffer at pH 8. Additionally, the buffer concentrations between 10 to100 mM concentration did not have any impact on the stability of theCTLA4-Ig composition at 2°-8° C. Similarly, the presence of sodiumchloride between 30 to 500 mM concentration had no effect on thesolution-state stability of the CTLA4-Ig composition stored at 2°-8° C.

Based on these results, the CTLA4-Ig composition at 10 mg/mL in 25 mMsodium phosphate buffer and 50 mM sodium chloride at pH 7.5 was selectedfor formulation at 50 mg/vial strength. The CTLA4-Ig concentration waslater changed to 50 mg/mL in the same buffer composition to allow forthe development of the CTLA4-Ig compositions with 200-and 250 mg/vialstrengths.

CTLA4-Ig for injection is formulated with maltose in addition to sodiumphosphate buffer and sodium chloride. The function of excipients used inthis product is listed in the Table above.

The presence of inorganic salts, such as sodium chloride and sodiumphosphate buffer components reduce the glass transition temperature(Tg′) of the frozen system. Moreover, dibasic sodium phosphate, which isformed in situ at pH 7.5 undergos preferential crystallization duringfreezing, which reduces the micro-environmental pH of the frozensolution. Based on these reasons, the minimum amounts of sodium chlorideand sodium phosphate buffer were selected to minimize their impact onthe lyophilization process. Maltose is added as a stabilizer which actsas a cryo-and lyo-protectant during lyophilization and upon subsequentstorage of the drug product, respectively. CTLA4-Ig composition forInjection, 250 mg/vial, is a sterile, single use vial withoutantimicrobial preservatives.

Example 3 Carbohydrate Content Analysis of a CTLA4-Ig Composition

The purpose of the method is to provide chromatographic profiles ofCTLA4-Ig N-linked oligosaccharides. This procedure can be used to obtainthe molar ratio of N-Acetyl Neuraminic Acid (NANA) and N-GlycolylNeuraminic Acid (NGNA) to protein in CTLA4-Ig samples (total bound plusfree). NANA and NGNA are two forms of sialic acid. The glycosylation onthe CTLA4-Ig protein contains N-linked oligosaccharides. For example,these oligosaccharides can be liberated by enzymatic hydrolysis withPNGase F over the course of 22 hours. The free oligosaccharides areprofiled using high pH anion exchange chromatography employingelectrochemical detection. Carbohydrate profiles for the N-linkedoligosaccharides were evaluated using High pH Anion ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD). Theseprofiles were confirmed and structural information was obtained usingPorous Graphitized Carbon (PGC) chromatography coupled with MS. Bothtechniques and results are described in this section. This procedure isexemplary for CTLA4-Ig of SEQ ID NO:

N-Linked Oligosaccharide Isolation: Cleavage of asparagine-linked(N-linked) oligosaccharides from CTLA4-Ig was performed by enzymatichydrolysis using PNGase F. To perform the deglycosylation, CTLA4-Ig wasfirst reduced and denatured: 1-2 mg of CTLA4-Ig in 176 μL of 5 mM sodiumphosphate buffer containing 0.5% SDS and 1% beta-mercaptoethanol washeated at 100° C. for 2 minutes, then allowed to cool to ambienttemperature. To the cooled mixture, 16 μL of 10% NP-40 were added. Thesample was mixed well and 40 μL of PNGase F (50,000 U/mL, in 50 mMsodium phosphate buffer) were added. The sample was mixed well andincubated at 38° C. for 24 hrs. The enzymatically-releasedoligosaccharides (glycans) were purified by reversed phase highperformance liquid chromatography (HPLC) using a Phenomenex Luna C18column (4.6×150 mm; 5 μL) coupled with a Thermo HyperCarb column(4.6×100 mm; 5 μL,) at a flow rate of 1.0 mL/minute; the chromatographused for the isolation was a Waters Alliance 2695 equipped with a Waters2996 detector. The columns were first equilibrated with 0.05%triflouroacetic acid (TFA). After injection of a sample, a gradient ofacetonitrile was initiated, terminating at 15 minutes with a solventcomposition of 0.05% TFA in 12% acetonitrile. The glycans were theneluted from the HyperCarb column with a step gradient to 0.05% TFA in60% acetonitrile. The glycans were collected while monitoring by UVabsorbance at 206 nm and were concentrated to dryness under vacuum.Prior to subsequent injections the Luna C18 column was cleaned with0.05% TFA in 40% acetonitrile, 40% isopropanol, 20% water.

Preparation of NANA Stock Solution (N-Acetyl Neuraminic Acid) (1 mg/mL).Important: Prior to weighing, allow the NANA standard to warm to roomtemperature. Failure to do so will result in water condensation in thestandard. Open only long enough to obtain desired amount, then resealbottle tightly and return to freezer, storing with desiccant. Accuratelyweigh between 3 and 10 mg of the N-Acetyl Neuraminic Acid. Record to thenearest 0.1 mg. Transfer to an appropriate size container if weighing isdone using weighing paper/boat. Add sufficient amount of HPLC gradewater to yield a concentration of lmg/mL solution. Mix with a stirringbar or by vortexing until dissolved. Store between 2 and 8° C. for up to3 months in a polypropylene tube.

Preparation of NGNA Stock Solution (N-Glycolyl Neuraminic Acid) (1mg/mL) Important: Prior to weighing, allow the NGNA standard to warm toroom temperature. Failure to do so will result in water condensation instock. Open only long enough to obtain desired amount then reseal bottletightly and return to freezer, storing with desiccant. Accurately weighbetween 3 and 10 mg (record to the nearest 0.1 mg of NglycolylNeuraminic Acid. Record to the nearest 0.1 mg. Transfer to anappropriate size container if weighing is done using weighingpaper/boat. Add an appropriate volume of HPLC grade water to yield atarget concentration of lmg/mL solution. Mix with a stirring bar or byvortexing until dissolved. Store between 2 and 8° C. for up to 3 monthsin a polypropylene tube.

System Suitability Solution. Add 0.050 mL each of 1 mg/mL of NANA andNGNA stock solutions to 0.900 mL HPLC grade water in an appropriatecontainer. Mix by vortexing. Store between 2 and 8° C. for up to 3months. N-Acetyl Neuraminic Acid Working Solution (0.050 mg/mL).Accurately measure 0.050 mL of 1 mg/mL NANA stock solution and add to0.950 mL of HPLC grade water. Mix by vortexing. Prepare in duplicate atthe time of use. N-Glycolyl Neuraminic Acid Working Solution (0.050mg/mL). Accurately measure 0.050 mL of 1 mg/mL NGNA stock solution andadd to 0.950 mL of HPLC grade water. Mix by vortexing. Prepare induplicate at the time of use.

Preparation of Samples and Hydrolysis Blank. Obtain the proteinconcentration for CTLA4-Ig material from the Certificate of Analysis(COA). Prepare a single sample of Hydrolysis Blank by adding 0.190 mL ofHPLC grade water to 1.5 mL micro centrifuge tube. Prepare two 1 mg/mLsolutions of samples and CTLA4-Ig Reference Material using HPLC gradewater. Mix by vortexing.

Hydrolysis of Samples, CTLA4-Ig Reference Material, and HydrolysisBlank. Perform the hydrolysis on duplicate preparations of referencematerial and samples in 1.5 mL micro centrifuge tubes. Note: It isimportant to use micro centrifuge tubes which will fit completely intothe heating block. Add 0.010 mL of 1 M H2SO4 to 0.190 mL of 1 mg/mLdilutions of samples and CTLA4-Ig reference material, and hydrolysisblank. Mix by vortexing and secure lid by lid-lock or tape. Place themicro centrifuge tubes in 80° C.±2° C. heating block for 1 hour ±2 min.After incubation, remove tubes from heating block, place tubes in microcentrifuge and spin to force sample to the bottom of the tube. Aliquothydrolyzed solutions into autosample vials and place in autosampler forinjection.

Instrument Conditions. Prepare the High Performance LiquidChromatography (HPLC) system. Set up the following conditions.Equilibrate the column for at least one hour at the flow rate of 0.6mL/min and a temperature of 40° C.

Mobile Phase(s) A: 5 mM H₂SO₄ B: HPLC Grade Water (Column Wash) FlowRate 0.6 mL/min Run Time 25 minutes Detector Wavelength 206 nm ColumnTemperature 40° C. Autosampler Temperature  4° C. Injection Volume 5 μLRetention Times NGNA 9.8 ± 1 minutes (system dependent) NANA 10.8 ± 1minutes (system dependent) Gradient conditions Isocratic

System Suitability. Start analysis with an injection of mobile phase asthe Instrument Blank to evaluate system baseline, which should be flatand stable. If the baseline is not flat and stable, additional blankinjections should be made. Note: A shift of the baseline should notexceed 0.25 AU. Perform six replicate injections of system suitabilitysolution. Calculate Resolution (R) and Theoretical Plates (N).

-   -   Using the first system suitability injection, calculate the        number of theoretical plates (N) using the following equation:

$\left. {{Number}\mspace{14mu} {of}\mspace{14mu} {Theoretical}\mspace{14mu} {Plates}}\rightarrow N \right. = {16\left( \frac{t}{W} \right)^{2}}$

-   -   Where:    -   N=Theoretical Plate Count    -   t=Retention time of NANA peak, in minutes    -   W=NANA Peak width at baseline, in minute

Calculate the moles of NANA and NGNA in the CTLA4-Ig Reference Materialand samples. NANA and NGNA standards are injected at the beginning andend of each run. Average the area counts for the replicate injections ofNANA and NGNA. Use this area in the following equation:

${{moles}\mspace{14mu} {of}\mspace{14mu} {NANA}\mspace{14mu} {or}\mspace{14mu} {NGNA}\mspace{14mu} {in}\mspace{14mu} {abatacept}\mspace{14mu} {reference}\mspace{14mu} {material}\mspace{14mu} {or}\mspace{14mu} {sample}} = \frac{(X)(Y)}{(Z)}$

-   -   X=Moles of NANA or NGNA in Working Solutions calculated in        Section 7.3.1    -   Y=Area counts of NANA or NGNA in abatacept reference material or        sample for each preparation (Note: see Section 7.2 and FIG. 3        for peaks to integrate for NANA)    -   Z=Averaged area of the replicate NANA or NGNA in Working        Solutions

In one embodiment, the resolution between the NGNA and NANA peaks mustbe >1.3. The theoretical plate count must be >or =to 4000. The systemsuitability injection reproducibility evaluated as % RSD of the areacounts of NANA peak must be ≤3%. The theoretical plate count must be≥4000. The system suitability injection reproducibility evaluated as %RSD of the area counts of NANA peak must be ≤3%.

N-Linked Oligosaccharide Profiling by High pH Anion ExchangeChromatography with Pulsed Amperometric Detection (HPAEC-PAD): HPAEC ofisolated oligosaccharides was performed on a chromatography systemconsisting of a Waters Alliance equipped with a Waters 464electrochemical detector utilizing a Dionex CarboPack PA1 (4×250 mm)anion exchange column and a Dionex CarboPack guard column. Theoligosaccharide samples were eluted using a sodium acetate gradient in200 mM sodium hydroxide (increasing sodium acetate concentration from 0mM at the time of injection to 225 mM at 60 minutes). Theelectrochemical detector was run under pulse mode with pulse potentialsE1=0.05V (t1=0.4 sec), S=0.75V (t₂=0.2 sec), E3=−0.15V (t₃=0.4 sec). Thedetection cell was composed of a gold working electrode, a stainlesssteel counter electrode and a silver/silver chloride referenceelectrode.

This method describes the procedure to determine the HPAEC (High pHAnion Exchange Chromatography) oligosaccharide profile of N-linkedoligosaccharides released from protein in CTLA4-Ig samples. The purposeof the method is to provide chromatographic profiles of CTLA4-Ig, suchas CTLA4-Ig drug substance N-linked oligosaccharides which can be usedfor comparative analysis between different compositions of CTLA4-Igmolecules. The glycosylation on the CTLA4-Ig protein contains N-linkedoligosaccharides. These oligosaccharides are liberated by enzymatichydrolysis with PNGase F (Peptide: N-Glycosidase F) over the course of22 hours. The free oligosaccharides are profiled using high pH anionexchange chromatography employing electrochemical detection.

CTLA4-Ig Bulk Drug Substance CTLA4-Ig in 25 mM Sodium Phosphate, 10 mMNaCl, pH = 7.5 Waters Total Recovery Vials with Waters Corporation,Catalog No. bonded PTFE/silicone septa 186000234 RapiGest SF WatersCorporation, Catalog No. 186001861 Alliance HPLC system equipped WatersCorporation with: Autosampler (refrigerated), Eluent Degas Module Model2465 Electrochemical Detector Column: CarboPac PA-1 4 × 250 mm DionexCorporation, Catalog No. 35391 Guard Column: CarboPac DionexCorporation, PA-1 4 × 50 mm Empower Catalog No. 43096 Data Collectionsystem

Oligosaccharide profiles of drug substance are evaluated againstconcurrently run samples of reference material. Results are reported aspercent deviation of selected domains and peaks from the same peaks inthe reference standards.

Chromatography Conditions for Oligosaccharide Profile by Anion-ExchangeChromatography

Column Temperature 29° C. Flow Rate 1 mL/min Mobile Phases and GradientGradient Program Conditions 1: 500 mM NaOAc 2: 400 mM NaOH 3: HPLC GradeWater Time (min) %1 %2 %3 Initial 0 30 70 0.0 0 30 70 11.0 0 30 70 12.04 30 66 20.0 10 30 60 80.0 45 30 25 81.0 0 30 70 100 0 30 70 Waters 2465settings Mode Pulse Empower settings Range = 5 μA E1 = +0.05 V E2 =+0.75 V E3 = −0.15 V t1 = 400 msec t2 = 200 msec t3 = 400 msec Samplingtime(ts) = 100 msec Time constant(filter)t = 0.1 sec Range offset = 5%Polarity + Temperature = 29° C. NOTE: Equilibrate the column anddetector with the initial mobile phase at the analysis flow rate forapproximately 2 hours, or until baseline is stable before makinginjections.

Autosampler Temperature set to: 4° C. Injection Volume 60 μL Run Time100 minutes Approximate Retention Times (RT; minutes) of dominant peaksin each Domain (see FIG. 1); values may vary depending on RT of SystemSuitability (SS) Standard Approximate RTs (min) SS: 18.5 Peak 1A: 20.0Peak 1B: 20.8 Peak 1C: 21.4 Peak 1D: 22.4 Peak 1E: 23.1 Peak 2 31.5 Peak3: 44.8 Peak 4: 58.5

Preparation of Mobile Phases for HPAEC Oligosaccharide CarbohydrateProfiling

HPAEC Eluent 1: 500 mM Sodium Acetate (NaOAc). Weigh 20.51±0.05 g ofSodium Acetate (anhydrous) into a 500 mL graduated cylinder containing400 mL of HPLC grade water. Bring volume to 500 mL with HPLC grade waterand stir for 5 minutes using a plastic serological pipette untilcompletely mixed. Filter the solution through a 0.2 μm nylon filter.Transfer to a 1 L eluent bottle. Cap the bottle loosely and sparge withhelium for 20 minutes. Tighten cap and pressurize the bottle withhelium. Store solution at room temperature under helium for up to threeweeks.

HPAEC Eluent 2: 400 mM Sodium Hydroxide (NaOH). Using a 1 L graduatedcylinder, measure 960 mL of HPLC grade water and transfer to a clean 1 Leluent bottle. Using a serological plastic pipet, add 40.0 mL of 10 NNaOH directly into the eluent bottle and mix the eluent by swirling. Capthe bottle loosely and sparge with helium for 20 minutes. Tighten capand pressurize the bottle with helium. Store solution at roomtemperature under helium for up to three weeks.

HPAEC Eluent 3: HPLC grade Water. Fill a 1 L eluent bottle withapproximately 1 L of HPLC grade water. Place eluent bottle on system,cap loosely, and sparge for approximately 20 minutes. Tighten cap andpressurize the bottle with helium. Store solution at room temperatureunder helium for up to three weeks.

50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH=7.5.

NaH₂PO₄•H₂O 6.9 g NaN₃ 0.2 g H₂O 1.0 liter final volume

Weigh out 6.9 g±0.1 g of NaH₂PO₄.H₂O and 0.2 g NaN₃ and dissolve in 800mL of HPLC grade H₂O in a 1 L reagent bottle using continuous mixingwith a magnetic stirring bar. Using a pH meter, adjust the pH of thesolution to 7.5 using 10M NaOH. Bring the final volume to 1.0 literusing a 1 L graduated cylinder. Store solution at room temperature forup to six months.

PNGase F Enzyme Working Stock in 50 mM Sodium Phosphate Buffer, 0.02%Sodium Azide, pH=7.5.

50 mM Sodium Phosphate Buffer 0.02% Sodium Azide, pH = 7.5. 1.8 mLPNGase F from Kit, Catalog No. P0704L 0.2 mL

Pipette 1.8 mL of 50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH7.5 into a 1.8 mL cryogenic vial. Add 0.2 mL of PNGase F from kit andmix thoroughly. Store solution at −20° C. or less for up to six months.The solution may be aliquoted prior to freezing.

External System Suitability Standard. Stachyose Stock Solution (1.25mg/mL). Weigh 0.125 g of Stachyose onto a weighing paper. Using ananalytical balance and transfer to a 100 mL volumetric flask. Fill tomark with HPLC grade water and mix thoroughly. Aliquot in 2 mL portionsinto Nalgene cryovials. Store solution at −20° C. or less for up to sixmonths.

Stachyose System Suitability Standard (12.5 μg/mL). Pipet 1 mL of the1.25 mg/mL stock into a 100 mL volumetric flask. Fill to mark with HPLCgrade water and mix thoroughly. Aliquot in 200 μL portions into 0.65 mLmicrofuge tubes. Place tubes in appropriately labeled storage box. Storesystem suitability solution at -20° C. or less for up to six months.

Standard and Sample Preparation

Reference Material Preparation. To a vial containing 1 mg of lyophilizedRapiGest SF, add 625 μL of 50 mM NaPhosphate buffer containing 0.02%NaAzide, pH 7.5. To a 0.65 mL Eppendorf tube add 120 μL of the RapiGestSF containing buffer. Add 40 μL of Reference Material (˜50 mg/mL). Thefinal RapiGest SF concentration should be 0.12% w/v. Add 40 μL of thePNGase F working stock, mix thoroughly, spin down the sample, and placeat 38±2° C. for 22±2 hours (water bath or the Alliance autosamplercompartment). Pipet sample into a microcon YM-10 centrifugal filter andcentrifuge at 13,000 g for 30 minutes. Place 200 μL of HPLC water in thefilter and rinse into the filtrate by centrifuging for an additional 30minutes at 13,000 g. Vortex the combined filtrate for 15 seconds andcentrifuge the sample for 10 seconds. Using a pipette transfer theresulting solution (˜380 μL) to an HPLC total recovery autosampler vial(item 1.15).

Sample Preparation. To a 0.65 mL Eppendorf tube add 120 μL of theRapiGest SF containing buffer. Add 40 μL of protein sample (this volumeshould equate to between 1 and 2 mg of CTLA4-Ig). The final RapiGest SFconcentration should be 0.12% w/v. Add 40 μL of the PNGase F workingstock mix thoroughly by vortexing for 10 seconds. Spin down the sample,and place at 38±2° C. for 22±2 hours (water bath or the Allianceautosampler compartment).Pipet sample into a microcon YM-10 centrifugalfilter and centrifuge at 13,000 g for 30 minutes. Place 200 μL of HPLCwater in the filter and rinse into the filtrate by centrifuging for anadditional 30 minutes at 13,000 g. Vortex the combined filtrate for 15seconds and centrifuge the sample for 10 seconds. Transfer the resultingsolution (˜380 uL) to a total recovery HPLC autosampler vial (item1.15).

System Suitability

Electrochemical Detector Cell Stabilization. Inject 30 μL of theexternal stachyose system suitability standard (12.5 μg/mL). Ensure thepeak height for stachyose is 800 nA. Ensure there is no excessiveelectrical noise from the cell and the baseline is flat. Arepresentative system suitability chromatogram is shown in FIG. 2. Ifthe stachyose sensitivity or the baseline is unacceptable, check thebuffer composition, clean the electrode or replace the electrode. Ifexcessive noise is present, check cell to ensure removal of all airbubbles. Restabilize the cell and re-inject stachyose standard.

Theoretical Plates (N). Determine the number of Theoretical Plates (N)based on the Stachyose peak using the formula below. This is donethrough the Empower data analysis system or may also be done manually.N=16 (t/W)² WHERE

-   -   t: retention time measured from time of injection to peak        elution time at maximum height    -   W: width of peak by extrapolation of sides to baseline.    -   N must be ≥6000. If the plate count is less than 6000, adjust        the run gradient or replace column.

Tailing Factor (T). Determine column Tailing Factor (T) based on theStachyose peak using the formula below. This is done through the Empowerdata analysis system or may also be done manually. T=(W₀₀₅/2f), WHERE:

-   -   W₀₀₅: width of peak at 5% of height (0.05 h).    -   f: the measurement (width) from front edge of peak at W₀₀₅ to        the apex of the peak.    -   T must be ≤1.2. If the tailing factor is greater than 1.2, check        buffer composition, replace the column or clean the column as        indicated in and re-inject system suitability standard.

Stachyose System Suitability Standard Retention Time Verification. Theretention time is system dependent. The stachyose system suitabilitystandard should exhibit a retention time of 18.5±2.0 minutes. CTLA4-IgStandard Material. Observe the carbohydrate profile from the firstbracketing reference material injected prior to injection of samples.The carbohydrate profile should be similar to that shown in FIG. 1.Absolute retention times are system dependent. Ensure that thedifference in retention times between the first peak in Domain I (Peak1A) and the main peak in Domain III (Peak 3) is between 22 minutes and28 minutes. If delineation of peaks does not resemble that obtained inFIG. 1 take appropriate actions (e.g. check instrument function, cleancolumn, check/replace buffers, replace column) and re-evaluate. Thefollowing procedure may be used to clean the column: turn off the celland clean the column with 80% Eluent 1, 20% Eluent 2 for 5 minutesfollowed by 50% Eluent 1, 50% Eluent 2 for 10 minutes. Re-equilibratethe column and cell (with cell turned on) at initial conditions andre-evaluate.

Injection Sequence

-   -   Set up the injection sequence of isolated oligosaccharides as        follows:    -   Stachyose Standard (30 μL)    -   Reference Material (60 μL)    -   Sample(s) (60 μL)    -   Reference Material (60 μL)    -   It is recommended that five samples be run between bracketing        reference material injections.

Data Analysis

Process the Chromatograms. Process the chromatograms for the ReferenceMaterial and samples in Empower. Set integration parameters so that peakdelineation and the baseline is similar to that shown in FIG. 75,integration lines may need to be placed manually. Perform calculationsfor relative Domain areas and relative peak areas shown. Determine theaverage values for these parameters for the CTLA4-Ig Material and foreach sample if replicate injections were made. For the ReferenceMaterial, determine relative deviation for Domains I, II, III, Peaks 1Aand 1B for each replicate with respect to the average of all replicates.

Comparison of Profiles of Sample to Reference Material Profiles. VisualComparison. Determine if both samples and Reference Material have thesame number of Domains and primary peaks. Primary peaks are those peakslabeled in FIG. 75 (Peaks 1A, 1B, 1C, 1D, 2, 3 and 4). RelativeQuantitation Comparison. Compare the relative areas of samples (DomainsI, II, and III and Peaks 1A, and 1B; if replicate injections were madeof samples use their average values) with the average relative areasfrom the bracketing CTLA4-Ig injections. Determine the relativedifference of these areas from the average CTLA4-Ig Material values.Calculations—% Domain Area (Relative Domain Area). Calculate the %Domain area for the Domains of the profiles for the Reference Materialand samples. Refer to FIG. 75 for pattern of Domain areas. Following theexample in FIG. 75, calculate the Domain percent ratios by using thefollowing information and formula (retention times are system dependentand reflect result in FIG. 75):

-   -   Domain I: Sum of the peak areas at approximate retention times        18-24 minutes (Peaks 1A-1E)    -   Domain II: Sum of the peaks from 26-38 minutes    -   Domain III: Sum of the peaks from 39-50 minutes    -   Domain IV: Sum of the peaks from 51-64 minutes    -   Domain V Sum of the peaks from 65-75 minutes    -   NOTE: Retention time windows for Domains will shift according to        variations in daily chromatographic performance. Adjust times        accordingly.

${{Domain}\mspace{14mu} {Area}\mspace{14mu} \%} = {\frac{{Individual}\mspace{14mu} {Domain}\mspace{14mu} {Area}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {Domain}\mspace{14mu} {Areas}} \times 100\%}$

-   -   For Domains I-III also calculate the average values in the        bracketing reference material injections, as well as in samples        if replicate injections are made.

% Peak Area (Relative Peak Area). Calculate the % peak area for Peaks1A, 1B, 1C, and 3 of the profiles for the Reference Material andsamples. Refer to FIG. 1 for pattern of peak areas; retention times aresystem dependent. Calculate the peak percent ratios by using thefollowing information and formula:

${{Individual}\mspace{14mu} {Peak}\mspace{14mu} {Area}\mspace{14mu} \%} = {\frac{{Individual}\mspace{14mu} {Peak}\mspace{14mu} {Area}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {Domain}\mspace{14mu} {Areas}} \times 100\%}$

For each of Peaks 1A and 1B, also calculate the average values in thebracketing reference material injections, as well as in samples ifreplicate injections are made. Calculation of the Percent Differencefrom Average Reference Material Values. Use the following formula tocalculate percent differences in average relative areas of DomainsI-III, Peaks 1A and 1B of samples compared to Reference Material:

% Diff=|RM−S|/RM×100

-   -   WHERE:    -   RM=average relative area value of interest for Reference        Material    -   S=average relative area value of interest for a sample    -   | |=absolute value

Exemplary Values. For a run to be acceptable the exemplary values mustbe met and all injections relevant to the sample must have successfullyoccurred. Additionally, for each of the bracketing Reference Materialinjections, the % Domain Areas for Domain I, II and III and % Peak Areasfor Peak 1A and 1B must be within 15% of their average values.

Analysis by HPAEC-PAD: N-linked oligosaccharides were cleaved fromCTLA4-Ig molecules and analyzed by HPAEC-PAD. Oligosaccharides eluteinto four domains based on the amount of sialic acid present. Domainswere established based on the migration of oligosaccharide standards andwere confirmed by MS. Domain I represents asialylated species. DomainsII, III, and IV represent mono-, di-, and tri-sialylated species,respectively. In order to characterize the structure of theoligosaccharides at the three N-linked sites, peptides T5, T7 and T14were individually isolated (refer to Table 59 for identity of thesepeptides). This was performed using tryptic digestion of CTLA4-Ig andmanually collecting peaks corresponding to these three peptides.

The isolated peptides were treated with PNGase F to release theoligosaccharides, which were subsequently purified and analyzed byHPAEC-PAD. Peptide T5 did not produce a good profile since it isdifficult to purify due to its extreme hydrophobicity. Quantities ofcleaved oligosaccharides are low due to low recovery of this peptidefrom the reversed phase chromatography step after tryptic digestion.

TABLE 59 Theoretically Expected and Observed Masses of CTLA4-Ig ExpectedObserved Fragment Residue Mass Mass No. No. (Daltons) (Daltons) PeptideSequence T1 + A −1-14 1536.8 1536.8 AMHVAQPAVVLASSR T1  1-14 1465.81465.7 MHVAQPAVVLASSR T2 15-28 1485.7 1485.6 GIASFVCEYASPGK T3 29-33574.6 574.2 ATEVR T4 34-38 586.7 586.3 VTVLR T5^(a) 39-83 4900.4 cQADSQVTEVCAATYMMGNELTFLD DSICTGTSSGNQVNLTIQGLR T6 84-93 1171.4 1171.4AMDTGLYICK T7^(a)  94-128 3997.5 c VELMYPPPYYLGIGNGTQIYVIDPEP CPDSDQEPKT8^(b) 129-132 435.2 ND SSDK T9^(b) 133-158 3345.7 cTHTSPPSPAPELLGGSSVFLFPPKPK T10 159-165 834.9 834.4 DTLMISR T11 166-1842139.3 2138.6 TPEVTCVVVDVSHEDPEVK T12 185-198 1677.8 1677.2FNWYVDGVEVHNAK T13 199-202 500.6 500.3 TKPR T14a 203-211 1189.2 cEEQYNSTYR T15 212-227 1808.1 1807.4 VVSVLTVLHQDWLNGK T16 228-230 438.5438.1 EYK T17 231-232 307.4 ND CK T18 233-236 446.5 ND VSNK T19 237-244838.0 837.4 ALPAPIEK T20 245-248 447.5 447.2 TISK T21 249-250 217.2 NDAK T22 251-254 456.2 456.2 GQPR T23 255-265 1286.4 1286.6 EPQVYTLPPSRT24 266-270 604.7 604.3 DELTK T25 271-280 1161.4 1160.5 NQVSLTCLVK T26281-302 2544.7 2545.6 GFYPSDIAVEWESNGQPENNYK T27 303-319 1874.1 1873.2TTPPVLDSDGSFFLYSK T28 320-324 574.7 574.2 LTVDK T29 325-326 261.3 ND SRT30 327-349 2803.1 2800.8 WQQGNVFSCSVMHEALHNHYTQK T31 350-356 659.7659.1 SLSLSPG T31 + K 350-357 787.9 787.0 SLSLSPGK T2 clip 15-23 NE1045.3 GIASFVCEY T12 clip 185-195 NE 1364.7 FNWYVDGVEVH T27 clip 303-317NE 1658.5 TTPPVLDSDGSFFLY T30 clip 327-335 NE 1125.3 WQQGNVFSC T30 clip327-333 NE 878.3 WQQGNVF ND not detected NE not expected from trypsindigestion (these masses were not expected from the digest, non-specificcleavage). ^(a)Tryptic peptides T5, T7, and T14 have N-linkedglycosylation. The mass listed is that of the peptide withoutglycosylation. ^(b)Tryptic peptides T8 and T9 have O-linkedglycosylation. The mass listed is that of the peptide withoutglycosylation. c Several different masses corresponding to differentglycoforms of glycosylated peptides were observed.

The HPAEC-PAD profiles for all N-linked carbohydrates from CTLA4-Ig andthe three peptides are shown in FIGS. 54A-54D. Panel A shows theN-linked oligosaccharide profile of the CTLA4-Ig molecule, while panelsB, C and D show the profiles for the T5, T7 and T14, respectively. Theamount of oligosaccharides injected for each of the peptides isdifferent due to the preparation processes. The majority ofoligosaccharides detected on peptide T5 consist of the mono-anddi-sialylated oligosaccharides. The profile of T7 contains primarilymono-, di-, and some a-and tri sialylated glycan species. Only a smallamount of the tri-sialylated structures can be detected on T5. PeptideT14 consists of predominantly asialylated oligosaccharides and a smallamount of mono-and di-sialylated oligosaccharides

The results obtained by HPAEC-PAD provide information on site-specificN-linked glycosylation. N-linked oligosaccharides from the CTLA4 regionof CTLA4-Ig contain a greater proportion of sialylated species thanthose from the Fc region of CTLA4-Ig.

Trypsin, Asp-N , and Trypsin/Chymotrypsin Peptide Mapping of CTLA4-Ig:CTLA4-Ig was denatured and reduced in 50 mM Tris buffer (pH 8.0)containing 6 M Guanidine and 5 mM dithiothreitol (DTT). After a 20minute incubation at 50° C., iodoacetamide (IAA) was added to a finalconcentration of 10 mM to alkylate free thiols, and the incubation wascontinued for an additional 20 minutes at 50° C. in the dark. Thereduced and alkylated mixture was loaded onto a desalting column(Amersham NAP-5), then eluted into the void volume with either 50 mMTris, 10 mM CaC12, pH 8.0 or 50 mM sodium phosphate buffer, pH 7.5.Following desalting, reduced/alkylated CTLA4-Ig was digested using twodifferent proteases: trypsin or Asp-N. For trypsin plus chymotrypsindigestion CTLA4-Ig is neither reduced nor alkylated.

For trypsin digestion, sequence grade trypsin (Roche, 2%, w/w,enzyme/protein) was added and incubated for 4 hours at 37° C. For Asp-Ndigestion, sequence grade Asp-N (Roche, 4%, w/w, enzyme/ protein) wasadded and incubated for 16 hours at 37° C. For the trypsin/chymotrypsindigestion, sequence grade trypsin (Promega, 4%, w/w, enzyme/protein) wasadded and incubated for 4 hours at 37° C., then a-chymotrypsin was added(Sigma, 4%, w/w, enzyme/protein) and incubated for 16 hours at 37° C.All samples were placed in a freezer after the digestion.

The resulting peptide mixtures were separated by gradient elution from aWaters Atlantis™ dC18 column (2.1×250 mm) on a Waters Alliance HPLCWorkstation at 0.120 mL/minute. The column was directly connected to theWaters Micromass Q-Tof micro™ mass spectrometer equipped with anion-spray source for collection of mass spectra. Peptide mixtures werealso separated on a Varian Polaris C18 column (4.6×250 mm) at 0.7mL/minute using the same HPLC workstation. The columns were equilibratedwith solvent A (0.02% TFA in water) and peptides were eluted byincreasing concentration of solvent B (95% acetonitrile/0.02% TFA inwater). A post-column splitter valve was used to direct 15% of the flowto the Q-Tof workstation, which was run in the positive TOF mode (m/z100-2000). The typical ion spray voltage used was 3000 V.

CTLA4-Ig Analysis by MS: CTLA4-Ig was diluted with 100 mM Tris, 25 mMNaCl, pH 8 to a final concentration of 0.7 mg/mL. PNGase F (New EnglandBiolabs) was diluted 30-fold with 100 mM Tris, 25 mM NaCl, pH 8 to afinal concentration of 17 U/μL. Equal volumes (60 μL each) of dilutedpurified fermentation sample and diluted glycosidase solution were mixedand incubated at 37° C. for 4 hours.

The resulting deglycosylated CTLA4-Ig (2 μg) was loaded onto apolymeric-based (copolymer of polystyrene and polyN-vinyl-2-pyrrolidinone) Waters Oasis® reversed phase extractioncartridge column (2.1×20 mm). The loaded column was washed with 5%solvent B (solvent A: 1% formic acid in water, solvent B: 1% formic acidin acetonitrile) at a flow rate of 0.2 mL/minute for five minutes todesalt, with the eluent diverted to waste. At the end of 5 minutes, afast gradient (5% solvent B to 95% solvent B in 10 minutes) began theelution of CTLA4-Ig off the column; here the eluent was directed intothe mass spectrometer (Waters Micromass Q-Tof micro™) at 45 μL/min afterflow splitting (chromatography system used was a Waters Alliance 2695equipped with a Waters 2996 detector).

The capillary voltage for the Q-Tof micro™ was set at 3 kV and thesample cone voltage at 40 V. The scans in every 0.9 second were averagedinto one scan; the inter-scan time was 0.1 second. The Q-Tof analyzerscans from m/z 800 to 2500. Spectra corresponding to the portion higherthan half the maximum peak height (in TIC chromatogram) are combinedusing Waters MassLynx™ software. The combined spectrum was subjected toWaters MaxEntl deconvolution. The resolution was set at 1 Da/Channel,and the uniform Gaussian damage model was selected with width at halfheight set between 0.5-1 Da. Minimum intensity ratios for the left peakand the right peak were both set at 50%.

N-Linked Oligosaccharide Analysis by Liquid Chromatography/MassSpectrometry (LC/MS) Using a Porous Graphitized Carbon (PGC): Isolatedoligosaccharides were separated on a porous graphitized column (ThermoHypercarb; 4.6×100 mm) using a Waters Alliance 2695 HPLC system equippedwith a Waters 2996 photodiode array detector. Oligosaccharide separationwas achieved using a two-stage gradient of increasing proportions ofacetonitrile in 0.05% TFA. In the first stage of the gradient, theacetonitrile percentage ranged from 9.6% at the time of injection to 24%at 80 minutes. In the second stage of the gradient, the acetonitrilepercentage ranged from 24% at 80 minutes to 60% at 110 minutes. A flowrate of 0.2 mL/minute was used throughout. The elution stream wasmonitored at by UV detection at 206 nm and analyzed by mass spectrometryusing a Waters MicroMass Q-Tof micro™ for mass identification.

Carbohydrate Analysis by LC/MS PGC method: Oligosaccharide isolation bydeglycosylation of the protein was performed by enzymatic hydrolysisusing PNGase F (New England Biolabs, Beverly, Mass.). For thedeglycosylation, between 1 and 2 mg glycoprotein in 160 μL of 50 mMsodium phosphate buffer containing 0.15% (w/v) Rapigest SF (WatersCorporation) was denatured by heating at 100° C. for 2 minutes. Thecooled solution was mixed and 40 μL of PNGase F (50,000 U/mL, in 50 mMsodium phosphate buffer, pH 7.5) was added. The sample was vortexedfollowed by incubation at 38° C. for 24 hours. Theenzymatically-released oligosaccharides were purified by highperformance liquid chromatography. Reversed phase liquid chromatographywas performed on a Phenomenex Luna 5 μL C18 column (4.6×150 mm,Phenomenex, Torrance, Calif.) coupled with a Thermo HyperCarb 5 μL(4.6×100 mm, Phenomenex, Torrance, Calif.) at a flow rate of 1.0 mL/min.The columns were equilibrated with 0.05% triflouroacetic acid (TFA)prior to injection. After sample injection (150 μL of the digestmixture), a gradient of acetonitrile was initiated terminating at 15minutes and a solvent composition of 0.05% TFA in 12% acetonitrile. Theglycans were then eluted from the HyperCarb column by washing with 0.05%TFA in 60% acetonitrile. The glycans were detected by UV absorbance at206 nm. Peaks eluted from the Hypercarb wash were collected andconcentrated to dryness under vacuum. Prior to subsequent injections theLuna C18 column was cleaned with 0.05% TFA in 40% acetonitrile, 40%isopropanol, 20% water.

Profiling of Isolated Oligosaccharides with PGC

The system used for PGC chromatography of isolated oligosaccharidesconsisted of a Waters Alliance equipped with a Waters 2996 photodiodearray detector utilizing a Hypercarb column (2.1×100 mm). Theoligosaccharide samples were eluted using an acetonitrile gradient.

Acidic Mobile Phase Elution: Acetonitrile gradient in 0.05%triflouroacetic acid (TFA). A two-stage gradient of increasingacetonitrile was used for the chromatographic separation ofoligosaccharides. The initial linear gradient of increasing acetonitrilevolume percentage from 9.6% at the time of injection to 24% at 80minutes is followed by a second gradient of increasing acetonitrilevolume percentage from 24% at 80 minutes to 60% acetonitrile at 110minutes. A flow rate of 0.15 ml/min was used throughout the gradient.The elution stream was monitored at 206 nm with a Waters UV detector,followed by a Micromass Q-Tof Micro for mass identification. Theionization parameters for the ESI probe were set as follows: Capillaryvoltage=3 kV, Cone voltage=45 V, Source temperature 80° C., anddesolvation temperature of 175° C.

Basic Mobile Phase Elution: An acetonitrile gradient in 0.4% ammoniumhydroxide (NH₄OH) was used for the chromatographic separation ofoligosaccharides. A linear gradient of increasing acetonitrile volumepercentage from 10.4% at the time of injection to 28% at 150 minutes ata flow rate of 0.15 mL/min was used to produce the profile. The elutionstream was monitored at 206 nm with a Waters UV detector, followed by aMicromass Q-Tof Micro for mass identification. The ionization parametersfor the ESI probe were set as follows: Capillary voltage=3 kV, Conevoltage=45 V, Source temperature 80° C., and desolvation temperature of175° C.

Direct profiling of oligosaccharide digest mixtures with PGC: The systemused for PGC chromatography of oligosaccharide digest mixtures consistedof a Waters Alliance fitted with both a Luna C18 and a Hypercarb porousgraphite column (4.6×100 mm, Thermo). The system was interfaced with aWaters 2996 photodiode array detector and a Q-ToF Micro (Micromass).Deglycosylation of protein was performed by enzymatic hydrolysis usingPNGase F. For the deglycosylation , between 1 and 2 mg glycoprotein in160 μL of 50 mM sodium phosphate buffer containing 0.15% by weightRapigest SF (Waters Corporation), was denatured by heating at 100° C.for 2 minutes. The cooled solution was mixed well and 40 μL of PNGase F(50,000 U/mL, in 50 mM sodium phosphate buffer, pH 7.5) was added. Thesample was mixed and then incubated at 38° C. for 24 hrs. Theenzymatically-released oligosaccharides were profiled by highperformance liquid chromatography. Reversed phase liquid chromatographywas performed on a Phenomenex Luna 5|u C18 column (4.6×150 mm) coupledwith a Thermo HyperCarb column 5 μL (4.6×100 mm) at a flow rate of 1.0mL/min. The columns were equilibrated with 0.05% triflouroacetic acid(TFA). After sample injection (150 of digest mixture) a gradient ofacetonitrile was initiated, terminating at 11 minutes and a solventcomposition of 0.05% TFA in 9% acetonitrile. A column switch was used toisolate the hypercarb column and the glycans are then eluted from theHyperCarb column with a linear gradient of increasing acetonitrilepercentage. An initial gradient of increasing acetonitrile percentagefrom 9% at the time of injection to 36% at 160 minutes was used. Thesecond gradient involved increasing acetonitrile volume percentage from36% at 160 minutes to 60% acetonitrile at 170 minutes. A flow rate of0.15 mL/min was used throughout the elution gradients. The glycans weredetected by UV absorbance at 206 nm, and by MS scanning the mass rangefrom 400-3000 m/z. MS parameters were set to the following values:Capillary 3 kV, Cone 45 V. Prior to subsequent injections the Luna C18column was cleaned with 0.05% TFA in 40% acetonitrile, 40% isopropanol,20% water.

Analysis by Porous Graphitized Carbon Chromatography: The structures ofthe oligosaccharides from Domains I-IV were investigated using PorousGraphitized Carbon chromatography (PGC) coupled to MS. N-linkedoligosaccharides were isolated from CTLA4-Ig and purified as describedin the previous HPAEC-PAD section. The oligosaccharides were analyzedusing a Hypercarb (PGC) column with a UV detector followed by Q-TOFESI/MS. The PGC profile for the N-linked oligosaccharides released fromCTLA4-Ig by PNGase F digestion is shown in FIG. 55 with domains noted.The order of elution is the same as that observed in HPAEC-PAD. Thestructures obtained are shown in FIG. 88A-88C with the nomenclature forthe oligosaccharides shown in FIG. 87.

In Domain I, six peaks were identified, corresponding to threeasialylated oligosaccharides (structures P2100, P2110, P2120). The 113Dalton mass difference between the predicted structure and observed massis due to detection of a trifluoroacetic acid (TFA) adduct. Differentpeaks with the same mass of 1,463 corresponding to P2100, indicates theyare likely different anomers.

In Domain II, six peaks were identified, corresponding to threebiantennary and triantennary oligosaccharides (structures P2111, P2121,and P3131, respectively), each containing one sialic acid residue(N-acetylneuraminic acid, NANA).

In Domain III, six peaks were identified, corresponding to threebiantennary, triantennary and tetraantennary oligosaccharides(structures P2122, P3132, and P4142, respectively), each containing twosialic acid residues (NANA).

In Domain IV, two peaks were identified, corresponding to twotriantennary and tetraantennary oligosaccharides (structures P3133 andP4133), each containing three sialic acid residues (NANA).

Measurement of molar ratio of moles sialic acid to moles CTLA4-Igmolecules or dimer by acid hydrolysis treatment of CTLA4-Ig molecules(see FIGS. 24 and 25): In this method, NANA and NGNA are cleaved fromthe protein by acid hydrolysis. The released NANA and NGNA are separatedby HPLC on a Rezex Monosaccharide RHM column and detected by UVabsorbance (206 nm). NANA and NGNA are quantitated based on the responsefactors of concurrently run NANA and NGNA standards. The test resultsare reported as molar ratios (MR) of NANA and NGNA respectively, toprotein. This assay determines the total number of moles, bound andunbound, of sialic acid.

Reagent, instrumentation and chromatographic conditions used in theassay: 1M sulphuric acid H₂SO₄ (stock) and 5 mM H₂SO₄ mobile phaserunning buffer; NANA standard solution of lmg/ml; NGNA standard solutionof lmg/ml. Alliance chromatographic system—Waters Corporation 2695Separations Module with integrated autosampler; Rezex monosaccharide RHMcolumn—8micrometer, 7.8×300 mm, Phenomenex, equipped with 7.8×50 mmguard column, Phenomenex; Detector—Waters Corporation 996 photodiodearray detector or Waters Corporation 2487 dual wavelength absorbancedetector. Chromatographic parameters: Flow—0.600 mL/min; Mobile phase—5mM H₂SO₄; Injection volume—5microL; Target concentration—1 mg/ml; Runtime—25 min; Column temperature—40° C.; Autosampler temperature—4° C.;Wavelength—206 nm; Retention time NANA (system dependent)—10.8 min (+ or−1 min), Retention time NGNA (system dependent) −9.8 min (+ or −1 min.).

System Suitability Standard: Add 50 μL each of 1 mg/mL of NANA and NGNAto 900 μL H₂O in an appropriate container. Store at 4° C. for up to 3months; N-Acetyl Neuraminic Acid Working Standard (0.05 mg/mL);N-Glycolyl Neuraminic Acid Working Standard (0.05 mg/mL).

Hydrolysis of Samples and CTLA4-Ig standard material: Samples andCTLA4-Ig standard material are diluted to 1 mg/mL in H₂O for hydrolysis.CTLA4-Ig samples and CTLA4-Ig standard material are hydrolyzed by adding10 μL 1 M H₂SO₄ to 190 μL of 1 mg/mL diluted samples and CTLA4-Ig.Hydrolysis is performed in duplicate in 1.5 mL micro centrifuge tubes.Lids are secured by lid-lock or tape. Tubes are mixed by vortexing andplaced in 80° C. heating block for 1 h. After incubation, tubes areremoved from the heating block, cooled down at room temperature for 3min, and placed in centrifuge for a quick spin to collect sample to thebottom of the tube. From the tubes, 50 μL are aliquoted of thehydrolyzed solution into sample vial, which is placed in cooledautosampler for injection. A hydrolysis blank is made as a singlepreparation by adding 10 μL 1 M H₂SO₄ to 190 μL water in 1.5 mL microcentrifuge tubes. The blank is processed as the samples.

System Suitability: To check the system suitability six replicateinjections of the system suitability standard (5 μL each) were injectedfollowed by one injection of the hydrolysis blank (5 μL). Using the lastsystem suitability standard injection, Resolution (R), acceptable valuesare higher than 1.3, and Theoretical Plates (N), accepatable theoreticalplate count must be at least 4000, were calculate respectively. Usingthe last five system suitability replicates, reproducibility of NANAcounts were calculated, and the hydrolysis blank was evaluated.

Resolution: Using the last system suitability standard injection, peakResolution was calculated using the following equation:R=2(T2−T1)/(W2+W1), where R is resolution, T2 is the retention time ofthe NANA peak (peak 2), T1 is the retention time of the NGNA peak (peak1), W2 is the width at the baseline of lines drawn tangent to the sidesof peak 2, W1 is the width at the baseline of lines drawn tangent to thesides of peak 1. FIG. 24 depicts a typical system suitability injection.

Theoretical plates (N): Using the last system suitability standardinjection, the theoretical plate count (N) was calculated using thefollowing equation: N=16(RT²/W), where N is the theoretical plate count,RT is the retention time of the NANA peak in minutes, W is the width atthe baseline of lines drawn tangent to the sides of the NANA peak.

The last five injections of the system suitability standard were used tocalculate the average area counts and their standard deviation for NANA.The relative standard deviation was equal or less than 3%. Thehydrolysis blank was free of any significant peaks with the retentiontime of NANA and NGNA.

Injection sequence: One injection each of the NANA and NGNA workingstandards was injected, followed by the hydrolyzed CTLA4-Ig samplematerial (duplicate samples), followed by the hydrolyzed CTLA4-Igmaterial. After the CTLA4-Ig runs were completed, one injection each ofthe NANA and NGNA working standards was injected.

To determine the moles of NANA or NGNA injected in the working standardthe following equation is used: mole NANA or NGNA=(C)(P)(I)/MW, where Cis the concentration of NANA and NGNA in the working standard, P is thepurity of the standard, I is the injection volume, MW is the molecularweight (309.2 g/mole for NANA, and 325.3 g/mole for NGNA).

To determine the moles of NANA and NGNA in the CTLA4-Ig samples, thefollowing equation is used: moles NANA or NGNA in sample=(X)(Y)/Z, whereX is the number of moles in the working standard of NANA and NGNA, Y isthe average counts of NANA and NGNA in the CTLA4-Ig sample, Z is theaveraged area of the duplicate NANA and NGNA in Working Standards. Fromthe duplicate injections of the standards, the area counts of the NANAand NGNA standard must have less than 10% RSD.

To determine the amount injected in each sample, the following equationis used: moles protein=(C)(D)(I)/MW, where C is the concentration ofCTLA4-Ig dimer in g/ml (obtained from UV analysis), D is the dilutionfor hydrolysis (0.95), I is the injection volume (0.00 ml) and MW is themolecular weight of CTLA4-Ig dimer as determined from mass spectrometry(92,439 g/mol).

Molar ratio (MR) of NANA or NGNA to CTLA4-Ig protein is calculated bythe following equation: MR=A/B, where A is the number of moles of NANAor NGNA, and B is the number of moles of CTLA4-Ig molecules or dimer.

Molar ratio (MR) of sialic acid, NANA and NGNA, to CTLA4-Ig protein iscalculated by the following equation: MR=(A+B)/C, where A is the numberof moles of NANA, B is the number of moles of NGNA, and C is the numberof moles of CTLA4-Ig molecules or dimer. Duplicates of CTLA4-Ig samplesand CTLA4-Ig standard material must have less than 10% RSD in molarratios for NANA.

Linearity of responses determined by hydrolysis method of measuringsialic acid content: NANA responses were demonstrated to be linear towith respect to NANA standard concentrations in the range from 0.5 μg/mL(˜0.1% nominal NANA standard=NANA˜QL) to 98.7 μg/mL (−200% nominal NANAstandard). NGNA responses were demonstrated to be linear to NGNAstandard concentrations in the range from 5.0 μg/mL (−10% nominal NGNAstandard) to 82.0 μg/mL (−160% nominal NGNA standard).

Responses of NANA from hydrolyzed CTLA4-Ig material are linear withrespect to protein concentrations in the range from 0.25 mg/mL (25%nominal CTLA4-Ig load) to 2.0 mg/mL CTLA4-Ig (200% nominal CTLA4-Igload). Responses of NGNA from hydrolyzed CTLA4-Ig material are linear toprotein concentrations in the range from 0.25 mg/mL (25% nominalCTLA4-Ig load) to 2.0 mg/mL CTLA4-Ig 200% nominal CTLA4-Ig load).

Accuracy of the hydrolysis method of measuring sialic acid content:Accuracy was demonstrated for CTLA4-Ig material (1 mg/mL) spiked withNANA or NGNA standards.

Precision of the hydrolysis method of measuring sialic acid content:Validation experiments demonstrated instrument precision (% RSD <3%),repeatability for sample preparations (% RSD <4%) and reproducibilityacross different sample preparations, different days, differentinstruments and different analysts (% RSD <6% for NANA, % RSD <12% forNGNA). NANA and NGNA molar ratios were considered to be precise within arange of 10% and 20%, respectively, of the reported results.

Range of the hydrolysis method of measuring sialic acid content: Theworking range for this assay was shown to be from 0.49 mg/mL to 3.87mg/mL CTLA4-Ig material.

Detection Limit (DL) of the hydrolysis method of measuring sialic acidcontent: The individual DL values for NANA and NGNA standards using aphotodiode array detector (PDA; HPLC System 1) were 0.464 μg/mL and0.402 μg/mL, respectively; the individual DL values for NANA and NGNAusing dual wavelength detector (HPLC system 2) were 0.131 μg/mL and0.111 μg/mL, respectively. The method DL, for both sialic species andbased on use of the least sensitive detector, was 0.5 μg/mL for NANA andNGNA.

Quantitation Limit (QL) of the hydrolysis method of measuring sialicacid content: The individual QL values for NANA and NGNA standards usinga photodiode array detector (PDA; HPLC System 1) were 1.68 μg/mL and1.52 μg/mL, respectively; the QL values for NANA and NGNA using a dualwavelength detector (HPLC System 2) were 0.48 μg/mL and 0.41 μg/mL,respectively. The method QL, for both sialic acid species and based onuse of the least sensitive detector, was 1.7 μg/mL for NANA and NGNA.

Ruggedness/Robustness: The method was demonstrated to be robust withrespect to the sample 48 hours refrigerated solution stability, the useof difference columns, the use of different NANA and NGNA lots and theuse of mobile phases with±5% alteration of concentration.

IEF gel electrophoresis: The pI of the glycoprotein can also bemeasured, before and after treatment with neuraminidase, to removesialic acids. An increase in pI following neuraminidase treatmentindicates the presence of sialic acids on the glycoprotein. An IEF gelcan be use to determine the isolecetrc point, the numer of isoforms ofCTLA4-Ig. A suitable system for running IEF gel is the Multiphore IIElectrophoresis System, and an IEF gel of pH 4.0 to 6.5 (AmershamBiosciences). The anode buffer has the following composition: 0.1MGlutamic Acid in 0.5M Phosphoric acid. The cathode buffer has thefollowing composition: 0.1M beta-Alanine. The IEF was gel is prefocusedunder constant power (25 watts) and current (25 mAmps) until the voltagereaches ≥300V. IEF calibration standards and samples of the appropriateconcentration were loaded and the gel was run under constant power (25watts) and current (25 mAmps) with maximum of 2000V for 2.5 hours. Afterfixing and Coomassie blue staining of the IEF gel, protein bands arevisualized by densitometer. A typical IEF gel of CTLA4-Ig dimerpreparation is shown in FIG. 10.

Example 4 Isolation and Characterization of Single Chain (Monomer) ofCTLA4-Ig

Preparation of native single chain CTLA4-Ig: Samples of CTLA4-Igrecombinant protein prepared by the methods of the invention wasseparated by non-denaturing SEC using a 2695 Alliance HPLC (Waters,Milford, Mass.) on two 21.5×300 mm TSK Gel® G3000SW_(XL) preparativecolumns (Tosoh Bioscience, Montgomery, Pa.) in tandem. Thirty injections(1.0 mL each) of the sample at ˜50 mg/mL were separated under isocraticconditions using a mobile phase consisting of 0.2 M NaH₂PO₄, 0.9% NaCl,pH 7.0, at a flow rate of 1.0 mL/min. Samples were monitored at anabsorbance of 280 nm using Water's 2996 PDA detector. Analysis wasperformed using Waters Millennium 4.0™ and Empower Pro© Software.Fractions were collected (1.0 mL each) on a Foxy 200 automated fractioncollector from 90 to 150 minutes. Fractions 16 to 39 (starting at 105 mLand ending at 129 mL) were pooled and concentrated using centriconconcentrators with a cutoff of 3500 MW.

The sample (2.0 mL at ˜4 mg/mL) was further chromatographed underdenaturing conditions using a HiLoad 26/60 Superdex 200 prep gradecolumn (Amersham Biosciences, Piscataway, N.J.) at an isocratic flowrate of 2.0 mL/min using 200 mM NaH₂PO4, 6.0 M guanidine hydrochlorideat pH 6.0 as mobile phase on an ÄKTAexplorer™ (Amersham Biosciences).Fractions 12-16 were collected, pooled, buffer-exchanged into 200 mMNaH₂PO₄, pH 6.0 using a HiPrep 26/10 desalting column (AmershamBiosciences), and finally concentrated.

Preparation of induced single chain CTLA4-Ig: Induced single chainCTLA4-Ig was prepared through denaturation, reduction, and alkylation ofCTLA4-Ig recombinant protein prepared by the methods of the invention.Guanidine hydrochloride (0.684 g) was weighed into a 1.5 mL Eppendorfcentrifuge tube, and 512 μL of 200 mM NaH₂PO₄, pH 6.0 was added andvortexed until the guanidine hydrochloride was completely dissolved.CTLA4-Ig recombinant protein was denatured by adding 238 μL of CTLA4-Igmaterial (concentration: 50 mg/mL) into the above tube and vortexed,resulting in a CTLA4-Ig final concentration of ˜10.0 mg/mL in 6.0 Mguanidine hydrochloride. The denatured protein was reduced by adding 2.6μL of 1.0 M DTT and incubating at 37° C. for 90 minutes. The reducedprotein was then alkylated by the addition of 0.047 g iodoacetamidesolid into the sample mixture, followed by vortexing, and incubation at37° C. for 60 minutes in the dark. The sample (2.0 mL at ˜4.0 mg/mL foreach injection) was chromatographed under denaturing conditions using aHiLoad 26/60 Superdex 200 prep grade column at an isocratic flow rate of2.0 mL/min using 200 mM NaH₂PO4, 6.0 M guanidine hydrochloride at pH 6.0on an ÄKTAexplorer™. The resulting single chain fractions (9-12) werecollected, pooled, buffer-exchanged into 200 mM NaH₂PO4, pH 6.0 on aHiPrep 26/10 desalting column, and concentrated.

MALDI-TOF mass spectrometry analysis of naive and induced single chainCTLA4-Ig: The single chain samples (20 μL) were desalted andconcentrated with C4 ZipTips (Millipore, Billerica, Mass.), then elutedwith 20 μL of 80% acetonitrile with 0.1% TFA saturated with sinnapinicacid. The mixture (1.0 μL) was spotted onto a well of the MALDI sampleplate and allowed to air dry before being placed in the massspectrometer. The MALDI-MS spectra were acquired on an OmniFlex massspectrometer (Bruker Daltonics, Mass.) using a nitrogen laser (337 nm).Samples were analyzed in the reflective, positive-ion mode by delayedextraction using an accelerating voltage of 20 kV and a delay time of200 ns. A total of 250 single-shot spectra were summed for each sample.External calibration was achieved using a mixture of Trypsinogen (23982m/z), Protein A (44613 m/z), and Bovine Albumin (66431 m/z).

Single Chain Analysis using Denaturing (Guanidine HCl) Size ExclusionChromatography: CTLA4-Ig supernatant from cell culture growth collectedat different points during the time course are prepared for HPLCanalysis. The samples are prepared by weighing 0.114 g guanidinehydrochloride (Mallinckrodt Baker Inc.) in a 0.65 mL Eppendorfmicrocentrifuge tube; adding 125 uL of time course CTLA4-Ig sample, andvortexing to completely dissolve the guanidine HCl. Then immediatelyadding 1.8 uL of 250 mM iodoacetamide and mix, and incubating at 37° C.for 30 minutes.

A tandem TSK-GEL®G3000SW_(XL) size exclusion column (7.8 mm ID×30 cm)with a TSK column guard (SW_(XL), 6.0 mm ID×4.0 cm) is used for thesingle chain SEC analysis performed on the Waters 2695 separationsmodule with a 2996 photodiode array detector. 25 μL of each sample isinjected onto the column equilibrated with 200 mM sodium phosphate, 6.0M guanidine hydrochloride pH 6.0 as mobile phase. The proteins areseparated on the column with a flow rate of 0.5 mL/min, and theresulting chromatogram is collected over a 60 minutes window. Theintegration and quantitation of individual peaks (monomer, single chain,etc.) are performed using the Empower Pro software. To ensure the HPLCsystem is working properly, injections are also made on the mobilephase, the protein sample buffer, the system suitability standard, andthe current CTLA4-Ig material before and after the sample injections.The peak resolution and plate count on the system suitability standardchromatogram are calculated.

Analysis of cysteinylation of CTLA4-Ig single chain by LC/MS PeptideAnalysis: CTLA4-Ig was denatured and reduced in 50 mM Tris buffer (pH8.0) containing 6 M Guanidine and 5 mM dithiothreitol (DTT). After a 20minute incubation at 50° C., iodoacetamide (IAM) was added to a finalconcentration of 10 mM to alkylate free thiols and the incubation wascontinued for an additional 20 minutes at 50° C. in the dark. Thereduced and alkylated mixture was loaded onto a desalting column(Amersham, NAP-5), then eluted into the void volume with either 50 mMTris, 10 mM CaCl2, pH 8.0 or 50 mM sodium phosphate buffer, pH 7.5.Following desalting, reduced/alkylated CTLA4-Ig was digested using twodifferent proteases: trypsin or Asp-N. CTLA4-Ig material was alsosubjected to trypsin/chymotrypsin digestion without reduction andalkylation.

For trypsin digestion, sequence grade trypsin (Promega, 2%, w/w,enzyme/protein) was added and the mixture was incubated for 4 hours at37° C. For Asp-N digestion, sequence grade Asp-N (Roche, 2%, w/w,enzyme/protein) was added and the mixture was incubated for 16 hours at37° C. For the trypsin/chymotrypsin digestion, sequence grade trypsin(Promega, 4%, w/w, enzyme/protein) was added and the mixture wasincubated for 4 hours at 37° C., then a-chymotrypsin was added (Sigma,4%, w/w, enzyme/protein) and the mixture was incubated for 16 hours at37° C. All samples were frozen (−20° C.) after the digestion.

The resulting peptide mixtures were separated by gradient elution from aWaters Atlantis^(TM) dC18 column (2.1×250 mm) on a Waters Alliance HPLCWorkstation at 0.12 mL/minute. The column was directly connected to theWaters Micromass Q-Tof micro™ mass spectrometer equipped with anion-spray source for collection of mass spectra. Peptide mixtures werealso separated on a Varian Polaris C18 column (4.6×250 mm) at 0.70mL/minute using the same HPLC workstation. The columns were equilibratedwith solvent A (0.02% TFA in water) and peptides were eluted byincreasing concentration of solvent B (95% acetonitrile/0.02% TFA inwater). A post-column splitter valve was used to direct 15% of the flowto the Q-Tof workstation, which was run in the positive TOF (time offlight) mode (m/z 100-2000). The typical ion spray voltage used was 3000V.

The loss of 113±4 u upon reduction suggests that there is a covalentdisulfide modification to the protein. The predicted shift forcysteinylation is 119.14 u. However, an actual mass loss of 111.14 u isexpected upon reduction of the single chain species. Loss of 119.14 uresults from the removal of a cysteine and a gain of 8 u results fromthe addition of 8 protons upon reduction of the eight intra chaincysteines (Cys^(47, 74, 92, 118, 197, 157, 303, 361) of SEQ ID NO:2).Thus, the additional 113±4 u on the intact mass corresponds tocysteinylation with a cysteine amino acid. The cysteine most likely tobe modified is Cys¹⁴⁶ since the interchain disulfide linkage is absentin the single chain species based on the intact MALDI data. Using LC/MSpeptide analysis to examine the peptides, which contain Cys¹⁴⁶, it isfound that reduced and non-reduced material display different retentiontimes and masses than the single chain material. To confirmcysteinylation, the single chain peptide containing Cys¹⁴⁶ was collectedand analyzed using MALDI.

The collected peak containing Cys¹⁴⁶ has a mass of 1787.48 u, inagreement with the predicted mass of 1787.51 u for the peptide with acysteinylation of Cys¹⁴⁶ (FIG. 26, panel A). This peptide, after beingsubjected to reduction, loses 119.11 u in agreement with a predictedloss of 119.14 u; the loss of cysteine (FIG. 26, panel B). The materialis then further manipulated with iodoacetamide producing a shift of56.99 u in agreement with a predicted mass gain of 57.03 u (FIG. 26,panel C).

Example 5 Manipulating Monomer or Multimer CTLA4-Ig Formation

Agonistic Effects of Media And Media Components on Single ChainFormation: The agonistic affects of different media and media componentsare determined for single chain formation. CTLA4-Ig molecules can beincubated at 37° C. with various media and individual media constituentsover a period of 60 hours and analyzed for single chain formation bytandem column denaturing SEC. An overwhelming agonist response forsingle chain formation is found upon the addition of 10 mM cysteine toformulation buffer. There is a rapid rise in single chain formationwhich peaks around six hours following the addition of cysteine. Thisresponse gradually decreases over the remaining 56 hours. In addition,the +30% gal feed affected a more gradual but still relatively rapidincrease in single chain formation. The +30% gal feed is a compositionof galactose and 117E. This mixture is added every day to feed thecells. While in this experiment, cysteine was introduced at artificiallylarge quantities independent of 117E, there is a need to determine whichof the 117E components can be involved in single chain formation.

Specific components of 117E and other media are incubated with CTLA4 Igover a period of 60 hours and analyzed for single chain formation bytandem column denaturing SEC. Investigation into this medium centersaround possible disulfide reducing components and/or inhibitors, whichwould effect single chain formation based on previous experimentsshowing the interchain disulfide is not present. The constituents of117E which are known to have some reducing affects on disulfides weretested; lipoic acid, cystine, cysteine, methionine, and glutathione.Again, an overwhelming agonist response for single chain formation isfound upon the addition of cysteine to formulation buffer. There is arapid response in single chain formation, which peaks at around sixhours, following the addition of cysteine. This response graduallydecreases over the remaining 56 hours. The major single chain formationoccurs with cysteine containing media: cysteine, yeastolates andfermentation media. The other sulfur containing components such asmethionine, and glutathione have no to very little affect on singlechain formation. There are no affects of ammonium chloride observed.

The present invention therefore encompasses a method for providing aratio of single chain: dimer form of a protein, such protein capable ofexisting in dimer as well as in single chain form, comprising the stepsof (1) providing and/or maintaining (such as during step (2)) a liquidcell culture medium for the culture of cells expressing said protein, inwhich the concentration of an agent capable of reducing or inhibitingdimer formation (such as cysteine) is selected to provide said ratio,and (2) culturing said cells to express said protein. Adding and/orincreasing the concentration of such an agent (for example, cysteine) ina liquid cell culture medium provides a higher ratio of singlechain:dimer form of such protein, while removing, decreasing oreliminating the concentration of such an agent (for example, cysteine)in a liquid cell culture medium decreases the ratio of singlechain:dimer form of said protein.

One particular embodiment of this method is where said protein is aglycoprotein capable of dimer formation through the formation of aninterchain disulfide bond, such as the CTLA4-Ig molecules of the presentinvention.

Agonistic Effects of High Salt on High Molecular Weight Formation:During the purification process, CTLA4-Ig is exposed to high saltconcentrations for varying amounts of time. The affects of high saltconcentrations are determined for high molecular weight formation.CTLA4-Ig (at ˜50 mg/mL) is incubated in the presence of 500 mM sodiumphosphate, pH=6.0, 37° C. There is an agonistic affect at highconcentrations of sodium phosphate; a sustained, rapid increase in highmolecular weight forms, mostly tetramer, is observed over a period of100 hours.

The present invention therefore encompasses a method for reducing theratio of aggregate: dimer form of a protein, such protein capable ofexisting in aggregate as well as in dimer form, during processing (suchas during purification) of such protein, comprising the use of one ormore liquids which are non-aggregate salt solutions. A “non-aggregatesalt solution” refers to a liquid containing a concentration of saltdissolved therein which is, relative to the same liquid containing ahigher concentration of such salt, less agonistic in the formation ofaggregate.

One particular embodiment of this method is where said protein is aglycoprotein capable of dimer formation through the formation of aninterchain disulfide bond, such as the CTLA4-Ig molecules of the presentinvention.

Antagonistic Effects on Single Chain Formation: The previous modelingexperiments demonstrated a large and rapid affect on single chainformation by the addition of cysteine containing components. Cysteine isan amino acid which contains a free sulfhydryl. If the sulfhydryl isinvolved in the formation of single chain it should be blocked throughthe use of antagonistic compounds. One such compound is iodoacetamide.Iodoacetamide is a water-soluble compound that reacts in a rapid fashionwith any free sulfhydryl to form an irreversible thioether bond.CTLA4-Ig is incubated at 37° C. with various medias, cysteine, andiodoacetamide over a period of 60 hours and analyzed for single chainformation by tandem column denaturing SEC and HMW by tandem columnnon-denaturing SEC. lodoacetimide not only blocks single chain formationbut also blocks aggregate formation in both a CTLA4-Ig composition andhigh salt. Iodoacetamide does not block the aggregate formation in lowsialic acid monomer. However, the amount of aggregate formed in lowsialic acid material is comparable to the amount formed in CTLA4-Igcomposition.

The model provides insight to a mechanism that has not previously beenwell understood. It appears that there are at least two major pathwaysof aggregate formation in the CTLA4-Ig process that have beenidentified. The first pathway, which produces the large amount ofaggregate, free sulfhydryl cysteine acts as an agonist for the formationof single chain species. The agonistic affect of cysteine can be blockedby the addition of iodoacetamide. Surprisingly, iodoacetamide is notonly an antagonist for single chain formation but also high molecularweight formation. It should be noted that the process is designed toproduce a composition that contains an increased amount of sialic acidas compared to fermentation during the downstream purification. In asecond path, which produces much less aggregate, a subspecies whichcontains low amounts of sialic acid is not affected by iodoacetamide forthe formation of single chain or aggregate.

The present invention therefore encompasses a method for decreasing theratio of single chain: dimer form of a protein, such protein capable ofexisting in dimer as well as in single chain form, comprising the stepsof (1) providing and/or maintaining (such as during step (2)) a liquidcell culture medium for the culture of cells expressing said protein,such medium containing an agent antagonistic to single chain formation(such as iodoacetamide), and (2) culturing said cells to express saidprotein.

The present invention also encompasses a method for decreasing the ratioof aggregate: dimer form of a protein, such protein capable of existingin aggregate as well as in dimer form, comprising the steps of (1)providing and/or maintaining (such as during step (2)) a liquid cellculture medium for the culture of cells expressing said protein, suchmedium containing an agent antagonistic to aggregate chain formationand/or antagonistic to single chain formation (such as iodoacetamide),and (2) culturing said cells to express said protein.

One particular embodiment of this method is where said protein is aglycoprotein capable of dimer formation through the formation of aninterchain disulfide bond, such as the CTLA4-Ig molecules of the presentinvention.

Based on these data, it is not difficult to imagine at least twopathways to aggregate formation are induced in CTLA4-Ig. In the majorpathway, single chain is involved in the formation of aggregate througha yet not completely clear mechanism. A second minor pathway, which isindependent of single chain formation, is involved in the formation ofaggregate. These pathways can help to explain why there are at leastthree forms of high molecular weight species that can bechromatographically separated. These are models and must be testedduring the actual fermentation process in order to determine the actualaffects if any and magnitude of the affects. Based on this dataconsideration should be given to testing not only the currentfermentation process but also fermentation devoid of cysteine.

Example 6 CTLA4-Ig Dimer and Tetramer CTLA4-Ig Dimer and TetramerBinding to B7-1 Ig

The invention provides methods for evaluation of CTLA4-Ig dimer andtetramer binding to B7-1Ig. Physical characteristics (e.g. diffusioncoefficient, molecular weight, binding valency) and instrumentoperational parameters (e.g. flow rate, chip density) can influence theBiacore assay results. Under mass transfer limitation; the binding rateof tetramer is approximately 20% slower than dimer for the same molarconcentration. At a specified flow rate, it is possible that tetramermolecules penetrated the matrix less efficiently compared to the dimer.Under a high density B7-1 Ig immobilized chip, competitive binding oftetramer and dimer exhibits comparable inhibition curves. This indicatesthat the theoretical valency of tetramer (four) versus dimer (two) hasno influence on binding to B7-1 Ig. Molar concentrations of tetramer canbe calculated based on a dimer standard curve. Using this approach, thebinding of tetramer to B7-1 Ig was found to have equivalent dosedependent response compared to dimer.

Comparison of the binding potency of a tetramer and a dimer isinfluenced by the unit of measurement used for the preparation ofstandards and samples. Tetramer and dimer samples can be compared at aconcentration of 2000 ng/mL. On a mass basis (ng/mL), each species showsa binding potency of approximately 100% using a standard curve of thesame species. However, the binding potency of tetramer was halved whendetermined on a dimer standard curve, and the binding potency of a dimerwas more than doubled when determined on a tetramer standard curve.Since the Biacore instrument detects molecular interactions based onmass, the signal resonance units (RU) from identical concentrations(ng/mL) of tetramer and dimer should be the same. Although the detectionsystem is a function of mass, the interaction between molecules occurson a molar basis. Therefore, the molar concentrations of CTLA4-Ig dimerand CTLA4-Ig tetramer are 21.6 nM and 10.8 nM, respectively, at 2000ng/mL. Using the molar concentration, both CTLA4-Ig dimer and CTLA4-Igtetramer samples show comparable binding potency on a dimer standardcurve. Using the same molar concentration approach on a CTLA4-Igtetramer standard curve, the CTLA4-Ig dimer sample shows an additional30% increase in binding potency compared to the tetramer sample. Thisobservation is due to a decrease in the slope of the tetramer standardcurve at high concentrations, resulting in a higher calculatedconcentration for a given initial binding rate. One explanation for thisobservation is the effect of mass transfer.

Mass transfer limitation experiment indicates that the initial bindingrate of the CTLA4-Ig tetramer is approximately 20% slower than theCTLA4-Ig dimer for the same number of molecules (i.e. the binding rateof the tetramer differs from dimer by a factor of 0.8). This observeddifference is due to the molecular weight of the two species and itseffect on diffusion of the molecules to the surface of the chip. Thediffusion coefficient of a molecule is inversely proportional to thecube-root of the molecular weight. A lower diffusion coefficient wouldindicate slower movement of the molecules. Based on respective molecularweights, the calculated diffusion coefficient of CTLA4-Ig tetramer is0.8 times that of the CTLA4-Ig dimer, or conversely the dimer is 1.25times that of the tetramer. Experimental data are consistent with thisobservation where the CTLA4-Ig dimer shows a potency of 133% ascalculated from a CTLA4-Ig tetramer standard curve using molarconcentrations. Mass transfer limitations on high-density chips are morepronounced at lower flow rates and lower analyte concentrations. As theflow rate increases, the dimer shows faster initial binding ratescompared to tetramer. Therefore, the increased molecular weight andlower diffusion coefficient of the tetramer contribute to initialbinding rate differences compared to dimer.

Competitive binding of CTLA4-Ig tetramer and dimer to B71Ig indicatesthat tetramer and dimer show similar binding valency under mass transferlimited conditions. The effects of additional tetramer onto a B7-1Igchip initially bound with either dimer or tetramer indicate low bindingpotential. This observation is not due to limited binding siteavailability because additional dimer could bind to B7-1Ig chipsinitially bound with CTLA4-Ig dimer or CTLA4-Ig teramer. Limitedpenetration into the matrix can explain the observed decrease intetramer binding.

The nature in which molecules bind on the Biacore affects theinterpretation of the results. The tetramer and dimer molecules diffuseat different rates due to their differences in molecular size under thecondition of mass transfer limitation. In addition, steric hindrance onthe surface of a high density chip affects penetration of subsequentmolecules to the matrix.

Physical characteristics such as the molecular weight, diffusioncoefficient, and binding of each species need to be considered whenperforming concentration analyses on the Biacore. Standards used forcomparison to the analyte of interest should be of the same material.However, tetramer can still be analyzed against dimer standards if boththe standards and samples are expressed on a molar basis where molecularsize is taken into consideration. The data presented indicate that thebinding of CTLA4-Ig dimer and CTLA4-Ig tetramer to B7-1Ig is comparable.

Both CTLA4-Ig dimer and tetramer show similar association rates(k_(on)). However, the tetramer shows a slower dissociation rate(k_(off)) which is attributed to avidity due to the increase in thenumber of binding sites.

Binding kinetic analysis between two proteins such as a ligand andreceptor can be performed on the Biacore using a chip immobilized with alow density of ligand of approximately 600-800 RUs such that the maximumbinding capacity (R_(ma)x) is in the range of 50-150 RU. The purpose ofa low-density chip is to minimize the effects of avidity and masstransport. Avidity is observed when multivalent analytes remain bound tothe surface of the chip due to close proximity of ligands available asdissociation of individual binding sites occurs. Mass transport isobserved when there is a significant difference in the analyteconcentrations between the surface of the chip and the bulk solution.

In one embodiment, the CTLA4-Ig molecule is a dimer consisting of twosingle-chain molecules linked by a single interchain-disulfide bond, andcontains two binding sites for B7 molecules. In another embodiment,formation of CTLA4-Ig tetramer, as confirmed by light-scattering, SECand SDS-PAGE, results in a molecule with potentially four binding sites.The binding kinetics of purified monomer and dimer are statisticallycompared to CTLA4-Ig dimer material. There are no significantdifferences in the k_(on) rates (p values >0.05). The k_(o)ff rate oftetramer is significantly different from the k_(o)ff rate of the dimer.The k_(off) rate of the dimer purified from the HIC cleaning peak is notsignificantly different from the k_(o)ff rate of the CTLA4-Ig dimermaterial. Therefore, the tetramer dissociates slower than the dimer,indicating an avidity effect due to the increased number of bindingsites per molecule.

Tetramer can be unfolded and dissociated into dimer by guanidinetreatment. This guanidine-treated CTLA4-Ig dimer was analyzed and theresults indicate that its binding kinetic characteristics were similarto those of the CTLA4-Ig dimer formed under physiological conditions.This observation supports the hypothesis that the observed difference inthe k_(o)ff rate between dimer and tetramer is related to bindingvalency of the molecules and the inherent nature of the specific Biacoremethod where avidity plays a role in the binding kinetics.

Affinity Purification of CTLA4-Ig Material from HIC Cleaning Peak:

Protein A-Sepharose affinity chromatography: 500 mL of HIC cleaning peakwas filtered through a 0.22 micron 1 L filter system (Corning, Corning,NY, Part no. 430517) and loaded onto a rProtein A-Sepharose column (5cm×15 cm), which was pre-equilibrated with phosphate buffered saline(Sigma, St. Louis, Mo., P-4417) at pH 7.4. The column was washed with700 mL PBS, pH 7.4 and eluted with 100 mM glycine, pH 3.5. Fractions of50 mL each were neutralized during collection by the addition of 0.5 mLof 2.0 M tris, pH 10 to the collection tubes. Fractions were assayed at280 nm absorbance, pooled and concentrated using a centriprep YM-3cartridge (Millipore Corporation, Bedford, Mass., Part no. 4203). Thepurified protein solution was stored at −70° C.

PROSEP-rA (recombinant Protein A) affinity chromatography: 500 mL of HICcleaning peak was filtered through a 0.22 micron 1 L filter system(Corning, Corning, NY, Part no. 430517) and loaded at a flow rate of 25mL/minute on a PROSEP-rA High Capacity (Millipore Corporation, Bedford,Mass.) column (25 mm×28 cm), which was pre-equilibrated with 25 mM Tris,pH 8.0 containing 250 mM sodium chloride. Waters PrepLC system equippedwith Waters 2767 Sample Manager and Waters 2996 Photodiode ArrayDetector was used for this chromatography. The column was washed with 25equilibration buffer for 30 minutes at a flow rate of 25 mL/minute andeluted with 100 mM acetate, pH 3.0 at a flow rate of 25 mL/minute for 30minutes. Fractions of 10 mL each were neutralized during elution bycollecting over 50 ul of 2.0 M tris, pH 10, which was previously addedto the tubes. Fractions having high absorbance at 280 nm were pooled andconcentrated using a centriprep YM-3 cartridge (Millopore Corporation,Bedford, Mass., Part No. 4203). The purified protein solution was storedat −70° C.

Size Exclusion Chromatography: Size exclusion chromatography wasperformed on a Waters Alliance 2695 separations module equipped with aWaters 2996 Photodiode Array Detector (Milford, Mass.), and a Foxy 200fraction collector controlled by Millennium³² version 3.20 or Empowersoftware. Tandem TOSOH BIOSCIENCE (Montgomery, Pa.) TSK G3000 SW (21.5mm×300 mm) and tandem TSK G3000 SWxL (7.8 mm×300 mm) columns were usedfor preparative and analytical SEC, respectively. Eluted proteins weremonitored by UV absorbance at 280 nm.

Preparative isolation of dimer, tetramer and multimer was achieved bySEC of Protein A purified HIC cleaning peak material. Thirteen samples(˜10 mg each) of Protein A eluate were injected onto a preparativetandem SEC column using a mobile phase of 100 mM monobasic sodiumphosphate buffer pH 7.0 containing 0.9% sodium chloride at a flow rateof 1 mL/min. Fractions were collected at every minute from 90-160minutes for each of the thirteen injections. The run time for a singleinjection was 180 minutes.

Each fraction was examined by tandem column analytical size exclusionHPLC. Fractions 13-15 (containing multimer), fractions 22 and 23(containing teramer), and fractions 43-49 (containing dimer) werepooled. Purified multimer, tetramer and dimer fractions were examined onanalytical two column SEC with dynamic light scattering detection (DSL).

Biospecific binding analysis of CTLA4-Ig component of the HIC cleaningpeak and purified components to immobilized B7-1 Ig: The biospecificbinding of CTLA4-Ig to immobilized B7-1Ig (on a CMS chip) was measuredusing a SPR based BIAcore C biosensor (BIAcore, AB, Piscataway NJ).CTLA4-Ig material was used to generate the standard curve. B7-1Ig wasimmobilized at a density of 5000 to 10,000 resonance units (RU's) on anactivated CMS sensor chip. CTLA4-Ig reference standards, qualitycontrols, and samples were injected at a flow rate of 20 μL/min. overthe B7-1 Ig sensor chip surface to generate sensorgrams. The initialbinding rate (RU/s) of CTLA4-Ig onto immobilized B7-1 Ig was measuredunder diffusion-limited conditions on a high density B7-1 Ig surface,and this correlates directly to the active concentration of CTLA4-Ig inthe samples. Standard, quality control sample, and unknown sampleconcentrations were interpolated from the standard curve generated byplotting the RU's versus CTLA4-Ig concentrations in the nominal range of125-8000 ng/mL. The final results were expressed as percent binding(Mean Concentration of unknown/sample/2000)×100.

Determination of molar mass and hydrodynamic radius: The SEC separationwas performed with a TSK3000 SWXL column and corresponding guard column.The mobile phase consisted of 25 mM HEPES, 850 mM NaCl, pH 7.0, usingisocratic conditions for elution at 0.8 mL/min. HPLC analyses wereperformed at ambient temperatures and samples maintained at 4° C. duringanalysis. Molar mass determination incorporated the Wyatt Dawn EOSutilizing 15 distinct scattering angles to measure the angular variationof light scatter for each species. A Zimm plotting formalism was usedfor molar mass determination where slices for each species were averagedfor molar mass. The specific refractive index increment (dn/dc) valueused to calculate absolute molar mass was 0.189 obtained using a) and anOptilab DSP Interferometer (RI. Hydrodynamic radius (Rh) determinationwas performed in-line with a Photon Correlator QELS detector positionedat an angle approximately 90° to the flow cell. The translationaldiffusion constant is measured from this signal and R_(h) is calculatedusing the Einstein-Stokes relationship. Data analysis was accomplishedusing Astra software version 4.90 from Wyatt Technology.

The molecular weight and hydrodynamic radius values for dimer andtetramer species were found to be 86-91 kDa and 172-199 kDa,respectively. The cleaning peak samples were observed to containadditional HMW species corresponding to hexamer and decamer by molecularweight. The range of the hydrodynamic radii for the dimer species is3.8-4.7 nm. The ranges of the hydrodynamic radii of the heterogeneoustetramer species were 5.7 nm to 6.2-6.3 nm.

Binding of CTLA4-Ig dimer and tetramer to B7-1 Ig Using Surface Plasmonresonance: Concentration Analyses: Concentration analyses of the variousCTLA4-Ig species to B7-1 Ig were performed on a Biacore 3000 instrument(Biacore, Piscataway, N.J.) according to Method 7441-4² with minormodifications. Modifications include the following: Biacore 3000 wasused instead of Biacore C. The flow rate was 10 μL/min instead of 20μL/min. Sample was injected for 60 seconds as opposed to 15 seconds. Thesensor chip surface was regenerated at 30 μL/min by three short30-second (15 μL) pulses of 10 mM sodium citrate, pH 4.0, containing 100mM NaCl (regeneration buffer), followed by one 30-second pulse of water.

A CMS sensor chip was immobilized with B7-1Ig at a concentration of 20μg/mL in acetate buffer, pH 5.0, aiming for a target density of6000-12000 resonance unit (RU). Standards were prepared by seriallydiluting CTLA4-Ig material to concentrations of 62.5-8000 ng/mL(0.675-86.3 nM) and dimer to concentrations of 125-16000 ng/mL(0.675-86.3 nM) in HBS-EP buffer. Test samples, consisting of eithermonomer or dimer, were diluted to a target concentration of ˜2000 ng/mLand analyzed on the Biacore. Concentrations were determined by theBIAevaluation software (version 4.0.1) using standard curves of eitherCTLA4-Ig material (>98% monomer) or purified dimer. The binding potencyis calculated as the percentage of the concentration determined on theBiacore divided by the concentration determined by A280 nm.

The binding rate of a molecule to a given surface is a function ofconcentration, which allows for the determination of unknownconcentrations. CTLA4-Ig dimer exhibits a higher binding rate as afunction of concentration (ng/mL) as compared to CTLA4-Ig tetramer.

The binding potencies of CTLA4-Ig dimer and tetramer are summarized inTable7. Based on ng/mL, the binding potency of a dimer sample iscalculated from a dimer standard curve and found to be 99.5%; whereas,an equivalent concentration of a tetramer sample is 47.2%. Conversely,the binding potency of a dimer sample is calculated from a tetramerstandard curve and found to be 266%; whereas, an equivalentconcentration of a tetramer sample is 103%. However, when dimer andtetramer are expressed as molar concentrations (nM), the bindingpotencies of both species are comparable. The binding potency of dimerand tetramer samples calculated from a dimer standard curve are 99.4%and 94.3%, respectively. On a tetramer standard curve, it is 133% and103%, respectively.

Standard curves of dimer and tetramer are comparable based on molarconcentrations.

TABLE 9 Binding Potencies of CTLA4-Ig Dimer and Tetramer. Dimer TetramerStandard Curve Standard Curve Sample ng/mL nM ng/mL nM Dimer 99.5% 99.4%266% 133% Tetramer 47.2% 94.3% 103% 103%

Binding Valency: CTLA4-Ig dimer (25-1600 nM) or tetramer (25-400 nM) waspre-mixed at various molar ratios with B7-1Ig for three minutes before30 (μL of the mixture was injected at a flow rate of 10 μL/min over aB7-1Ig chip immobilized with a density of 9392 RU. The chip wasregenerated after each injection by three 30-second pulses ofregeneration buffer followed by one 30-second pulse of water. The RU'sobtained at the end of each injection were compared.

Theoretically, the binding valency of the dimer molecule is two; eachsingle chain consists of one binding site. A tetramer molecule consistsof two dimer molecules, and thus has a binding valency of four. Todetermine the apparent binding valency of dimer and teramer, acompetitive assay was designed and conducted on the Biacore 3000. In theexperiment, analytes were pre-mixed with B7-1Ig at various molar ratiosfor three minutes before the mixture was injected onto a B7-1Ig chip(9392 RU) at a flow rate of 10 μL/min for one minute. Table 10 shows thepercentage of either dimer or tetramer that was competitively inhibitedwith increasing molar amounts of B7-1Ig. At a molar ratio of 1.25 (B7-1Ig) to 1 (dimer or tetramer), a significant difference was observed incompetitive inhibition with monomer at 96.1% and dimer at 84.9%.However, dimer and tetramer exhibit similar inhibition curves, thissuggests that the valencies are approximately equal. In addition, bothdimer and teramer also showed similar inhibition profiles using lowerdensity chips.

TABLE 10 Inhibition of Dimer and Tetramer with B7-1Ig. Dimer TetramerB7-1Ig (% Inhibition) (% Inhibition) Molar Ratio N Avg S.D. N Avg S.D.0.02 to 1 1 1.5 n/a 1 3.4 n/a 0.04 to 1 2 1.2 0.2 2 5.1 0.3 0.08 to 1 32.9 1.1 3 7.4 0.8 0.16 to 1 4 5.5 0.7 4 12.6 0.9 0.32 to 1 5 14.7 3.3 520.9 2.2 0.64 to 1 5 38.1 6.1 4 40.8 3.5 1.25 to 1 5 96.1 2.6 3 84.9 4.6 2.5 to 1 4 99.7 0.1 2 97.5 0.5  5.0 to 1 3 99.9 0.1 1 99.4 n/a 10.0 to1 2 100.0 0.0 n/a n/a n/a 20.0 to 1 1 100.0 n/a n/a n/a n/a

Saturation of B7-1Ig Chip: CTLA4-Ig dimer or tetramer (200, 1000, or8000 ng/mL) was initially injected at 10 μL/min for one minute over ahigh density B7-1Ig chip (6738 RU) followed by a series of seven1-minute injections with either monomer or dimer (200, 1000, or 8000ng/mL). The chip was regenerated after each condition by four 25 μLinjections of regeneration buffer followed by 25 μL injection of water.The RU's obtained at the end of each injection were compared.

Either dimer or tetramer was repeatedly injected over a B7-1Ig surfacepre-coated with either dimer or tetramer without surface regeneration.Initial binding with tetramer does not impede subsequent injections ofdimer from binding, however, additional tetramer injections result in asignificantly decreased rate of binding as compared to dimer injection.Initial binding with dimer followed by subsequent injections of dimerresults in an increased binding towards saturation. Subsequent injectionof tetramer to the dimer pre-coated chip shows a gradual decrease inbinding, indicating a dissociation of molecules from the chip and a lackof tetramer penetration into the matrix. Similar results are observedwith initial injection of either 200 ng/mL or 8000 ng/mL of CTLA4-Igmolecules.

CTLA4-Ig tetramer has higher avidity to the B7-1Ig receptor: CTLA4-Igspecies, including, the discarded portions of purification columns suchas the cleaning peaks of HIC and QFF columns were purified and theirbinding kinetics were analyzed on the Biacore.

CTLA4-Ig Species from HIC Cleaning Peak: The HIC column is used in theCTLA4-Ig process to remove high molecular weight species such as DNA.CTLA4-Ig species from the cleaning peak from the HIC column can be usedfor subsequent purification and kinetic analysis. The HIC cleaning peakwas passed through a Protein A column to capture all CTLA4-Ig speciesand to remove other impurities that can be present. The eluate from theProtein A column, which consisted of a mixture of all CTLA4-Ig species(i.e., dimer, tetramer, hexamer multimer), showed apparent k_(on) andk_(o)ff rates which were comparable to the CTLA4-Ig dimer standard.Separation of the Protein A eluate by 2-column SEC resulted in threeCTLA4-Ig species: dimer, tetramer, and hexamer/multimer, with k_(on) andk_(o)ff rates as summarized in Table 11. Sialic acid content of thepurified dimer from the HIC cleaning peak was low compared to CTLA4-Igdimer.

In Table 11, sample concentrations of 75-200 nM were tested on B7-1 Igchip (694 RU). CTLA4-Ig species purified from the HIC cleaning peakshowed lower binding compared to CTLA4-Ig reference. Tetramer which wasdisaggregated gave comparable k_(on) and k_(o)ff rates to CTLA4-Igreference.

TABLE 11 Kinetic Analysis of CTLA4-Ig Species from HIC Cleaning Peakk_(on) × 10⁵ k_(off) × 10³ K_(A) × 10⁷ K_(D) Species (1/Ms) (1/s) (1/M)(nM) CLTA4-Ig dimer standard 4.03 9.65 4.18 23.9 Protein A Eluate 3.779.73 3.87 25.8 Dimer 1.52 5.88 2.59 38.7 Tetramer 1.65 8.07 2.04 48.9Hexamer 1.54 12.3 1.25 79.9 Dimerized Tetramer 3.12 9.88 3.16 31.7

Statistical analysis of the data was performed using the Student'sT-test based on 7 observations of the CTLA4-Ig dimer standard and 14observations of purified dimer and tetramer. There was no difference inthe k_(on) rates comparing the CTLA4-Ig dimer standard with eitherpurified dimer or tetramer. However, the k_(o)ff rate and K_(D) werestatistically significant when the reference was compared with purifiedtetramer (Table 12). Comparing the purified tetramer to purified dimer,both the k_(on) and k_(o)ff rates as well as the K_(D) werestatistically different. It should be pointed out that although the datawere grouped into dimer and tetramer, individual classifications ofsamples (i.e., frontal, backside, etc) can have slightly differentcharacteristics that can affect their binding kinetics. For the dimer,the k_(on) and k_(off) rates averaged 3.3±1.0×10⁵M-1 s-¹ and8.8±3.5×10⁻3s-¹, respectively. The k_(on) and k_(off)rates of thetetramer were 2.6±0.8×10⁵M-1 s-¹ and 3.1±1.4×10⁻3s-¹, respectively.

In Table 12, Dimer (n=14) and tetramer (n=14) and CTLA4-Ig dimerstandard (n=7) were analyzed.

TABLE 12 Statistical Analysis of CLTA4-Ig Species. p-values Student'sT-test k_(on) k_(off) K_(D) Dimer standard vs. Dimer 0.9118 0.86780.7893 Dimer standard vs. 0.1044 0.0000002 0.0002 Tetramer Dimer vs.Tetramer 0.0372 0.000006 0.0004

CTLA4-Ig Species from QFF Cleaning Peak: The QFF column is the lastpurification column used to clean up residual impurities from theproduct. CTLA4-Ig species were isolated from the cleaning peak of theQFF column using the 2-column SEC method and analyzed on the Biacore3000. The data showed that the binding kinetics of this “QFF cleaningpeak” sample was similar to the CTLA4-Ig dimer standard. In addition,both dimer and tetramer purified from the QFF cleaning peak gave similarbinding kinetics compared to those purified from the composition.Furthermore, sialic acid contents of the purified monomer fractions weregreater than that of CTLA4-Ig dimer standard.

Guanidine Treatment (Dimerization of the tetramer): The tetramer can beconverted to dimer by treatment with guanidine followed by dialysis intophosphate buffer and confirmed to exist as a dimer by analytical SEC.Kinetic analysis of the “dimerized” tetramer from the HIC cleaning peakshowed that its k_(on) and k_(off) rates were similar to those ofCTLA4-Ig dimer.

Example 7 Intact Analysis by MS Electrospray Ionization (ESI)

CTLA4-Ig was diluted with 100 mM Tris, 25 mM NaCl, pH 8 to a finalconcentration of 0.7 mg/mL. PNGase F (New England Biolabs) was diluted30-fold with 100 mM Tris, 25 mM NaCl, pH 8 to a final concentration of17 units/μL. Equal volumes (60 μL each) of diluted sample and dilutedglycosidase solution were mixed and incubated at 37° C. for 4 hours.

The resulting deglycosylated CTLA4-Ig (2 μL) was loaded onto a WatersOasis® reversed phase extraction cartridge column (2.1×20 mm). Theloaded column was washed with 5% solvent B (solvent A:1% formic acid inwater, solvent B: 1% formic acid in acetonitrile) at a flow rate of 0.2mL/minute for five minutes to desalt, with the eluant diverted to waste.At the end of 5 minutes, a fast gradient (5% solvent B to 95% solvent Bin 10 minutes) began the elution of CTLA4-Ig off the column; here theeluant was directed into the mass spectrometer (Waters Micromass Q-Tofmicro™) at 45 μL/min after flow splitting (chromatography system usedwas a Waters Alliance 2695 equipped with a Waters 2996 detector).

The capillary voltage for the Q-Tof micro™ was set at 3 kV and thesample cone voltage at 40 V. The scans (every 0.9 second) were averagedinto one scan; the inter-scan time was 0.1 second. The Q-Tof analyzerscans from m/z 800 to 2500. Spectra corresponding to the portion higherthan half the maximum peak height (in TIC chromatogram) were combinedusing Waters MassLynx software. The combined spectra were subjected toWaters MaxEntl deconvolution. The resolution was set at 1 Da/Channel,and the uniform Gaussian damage model was selected with width at halfheight set between 0.5-1 Da. Minimum intensity ratios for the left peakand the right peak were both set at 50%.

Example 8 Matrix Assisted Laser Desorption Ionization-Time of Flight(MALDI-TOF)

MALDI-MS spectra were acquired on an OmniFlex™ (Bruker Daltonics, MA)using a nitrogen laser (337 nm). Protein samples were used withoutdesalting or desalted using solid phase extraction in the form of C4ZipTip® pipette tips (Millipore, Bedford Mass.). The pipette tips werewetted with acetonitrile-water (1:1 v/v) and equilibrated with 0.1%trifluoroacetic acid (TFA) prior to use. The pipette tips were thenloaded by drawing and expelling 10 μL of sample from the pipette threetimes. The loaded sample was washed three times with 10 μL of 0.1% TFA.Desalted protein samples were eluted from the pipette with 10 μL ofacetonitrile to water (1:1 v/v). Samples were spotted by mixing a 1 μLsample (either desalted or buffer containing) with 1 μL of matrixsolution and placing 1 μL of the mixture on the stainless steel target.The matrix solution was a saturated solution of sinapic acid in 1:1water to acetonitrile (v/v) containing 0.1% TFA. The mixture was spottedonto the MALDI sample plate and allowed to air dry before being placedin the mass spectrometer. All protein samples were analyzed in thelinear, positive-ion mode by delayed extraction using an acceleratingvoltage of 20 kV and a delay time of 200 nanoseconds. A total of 400single-shot spectra were accumulated from each sample. Externalcalibration was achieved using a mixture of standard proteins containingtrypsinogen (23982 m/z), Protein A (44613 m/z), and bovine albumin(66431 m/z).

MALDI-TOF MS Analysis of Peptides

Peptide mixture was separated by reversed phase chromatography andfractions from chromatographic peaks were collected and evaporated todryness. Sample was reconstituted in 50 μL of 25 mM phosphate buffer pH7.5. DTT was added to a final concentration of 5 mM and the fractionswere incubated at 50° C. for 20 minutes. After reduction, IAM was addedto a 10 mM final concentration and incubated in darkness at 50° C. foran additional 20 minutes.

MALDI-MS spectra were acquired on an OmniFlex (Bruker Daltonics, Mass.)using a nitrogen laser (337 nm). Samples were prepared by mixing a 1 μLsample with 1 μL of matrix solution. The matrix solution was a saturatedsolution of a-cyano-4-hydroxycinnamic acid in 1 to 1 water: acetonitrilewith 0.1% TFA. The mixture was spotted onto a well of the MALDI sampleplate and allowed to air dry before being placed in the massspectrometer. All peptides were analyzed in the reflective, positive-ionmode by delayed extraction using an accelerating voltage of 20 kV and adelay time of 200 nanoseconds. A total of 100 single-shot spectra wereaccumulated from each sample. External calibration was achieved using amixture of standard peptides containing angiotensin II (1046.54 m/z),angiotensin I (1296.68 m/z), substance P (1347.74 m/z), bombesin(1619.82 m/z), ACTH clip 1-17 (2093.09 m/z), ACTH clip 18-39 (2465.20m/z), and somatostatin (3147.47 m/z).

Example 9 Analysis of Media And Media Constituents Effects On CTLA4-IgSingle Chain and Multimer Species

CTLA4-Ig dimer, a low sialic acid sub-fraction, a high sialic acidsub-fraction, monomer frontal, and monomer species of CTLA4-Ig areprepared and purified. These samples are used in a series of modelingexperiments designed to determine the affects of media and mediaconstituents on single chain and high molecular weight formation overtimes between zero to at least 60 hours. The affects of formulationbuffer, iodoacetamide, sodium phosphate, ammonium chloride, basalmedium, fermentation broth, medium 177e, medium Concentrated AcidSolution I, medium concentrated acid solution II, insulin, EDTA,cysteine, lipoic acid, glutathione, methionine, and yeastolates aloneand in combinations are tested.

Example 10 Analyzing CTLA4-Ig Molecules by Size

A method of size exclusion chromatography (SEC) which uses denaturingconditions can be employed for the quantitation of protein species ofdifferent size. In one embodiment tandem SEC method using denaturingconditions can be employed for the quantitation of CTLA4-Ig single chainspecies. Single chain CTLA4-Ig can be a species lacking the inter-chaindisulfide bridge. Single chain CTLA4-Ig species isolated during thepurification process is referred to as native single chain CTLA4-Ig.Purified single chain produced by reduction and alkylation of CTLA4-Igdimer is referred as induced single chain. Native and induced singlechain CTLA4-Ig have the same characteristics.

Materials:

-   -   Potassium Phosphate Monobasic (KH₂PO₄) ACS grade    -   Potassium Hydroxide (KOH) 45% w/w Solution ACS grade    -   Sodium Chloride (NaCl) ACS grade    -   Calibrated Adjustable Single Pipettor, 100_L    -   Rainin, (Catalog No. P-100)    -   Water (H₂O) HPLC grade    -   2.0 mL Cryogenic Vials Nalgene, (Catalog No. 5000-0020)    -   Concentrated HydrochloricAcid (HC1) Fisher (Catalog No.        A144-212)    -   Sodium Hydroxide, (NaOH) 10N Solution    -   J. T. Baker, (Catalog No. 5674-02)    -   1000 mL Filter Unit 0.22 mm Corning, (Catalog No.430517)    -   Polypropylene 15 mL test tube Falcon, (Catalog No. 352097)    -   Sodium Azide (NaN3) ACS grade    -   Sodium Phosphate Monobasic, Monohydrate (NaH2PO4.H2O)    -   Potassium Hydroxide (KOH) Pellets

Instrumentation and Conditions

-   -   HPLC System Waters 2695 Separations Module    -   Column Toso Haas 5 μTSK 3000 SWXL, 300 mm×7.8 mm I.D. (Part No.        08541)    -   Guard Column Toso Haas 5 μTSK 3000 SWXL, 40 mm×6.0 mm I.D. (Part        No. 08543)    -   Detector Waters 2487 Dual Wavelength Detector Wavelength 280 nm        Flow Rate 1 mL/min    -   Integration System Empower    -   Injection Volume 20 μL    -   Assay Target Conc. 10 mg/mL    -   Mobile Phase 0.2 M KH2PO4, 0.9% NaCl, pH 6.8 with KOH    -   Assay Run Time 20 min    -   Column Temperature Ambient    -   Sample Temperature 4° C.    -   Retention Time Monomer 8.7 min±1.0 min, HMW species at 7.5        min±1.0 min, if present MW species will elute after the Monomer        peak.

Reagents

-   -   4N Potassium Hydroxide (4N KOH) (100 mL)    -   Use one of the following preparations as described:    -   Add 40 mL of HPLC grade water and 11.6 mL of 45% w/w solution of        KOH to a 100 mL volumetric flask. Bring volume up to 100 mL with        HPLC grade water.    -   In a 100 mL volumetric flask add 80 mL of HPLC grade water,        weigh 22.4 grams of KOH pellets, and stir magnetically until        completely dissolved. Bring to volume of 100 mL with HPLC grade        water.    -   Transfer solution into a 250 mL glass reagent bottle. Mix well        by invertion. Store at room temperature for up to 1 year.    -   Mobile Phase (0.2 M KH₂PO₄, 0.9% NaCl, pH 6.8)    -   Weigh out 27.2 grams of KH₂PO₄ and 9.0 grams of NaCl into a 1000        mL beaker.    -   Dissolve the solids in 800 mL of HPLC grade water using        continuous mixing with a magnetic stirring bar.    -   Using a pH meter, adjust the pH of the solution to 6.8 using 4N        KOH solution. If the pH exceeds 6.8, adjust it with concentrated        HCl.    -   Bring the final volume to 1 liter using a 1000 mL graduated        cylinder. Filter the solution through a 0.22 μm filter unit.    -   Transfer into a 1000 mL glass reagent bottle and sonicate while        vacuum degassing for 5+/−1 minute. Degas before each use.    -   Store at room temperature for up to 1 month.    -   2N Sodium Hydroxide (2N NaOH)    -   Transfer 20 mL of 10N NaOH into a 100 mL glass graduated        cylinder.    -   Bring volume up to 100 mL with HPLC water.    -   Transfer solution into a 250 mL glass reagent bottle. Mix well        by invertion. Store at room temperature for up to 1 year.    -   Weigh 3.45 grams of NaH₂PO₄.H₂O and 2.92 grams of NaCl and        dissolve with mixing with a stir bar in 900 mL of HPLC grade        water. Using a pH meter, adjust the pH of the solution to 7.4        with 2N NaOH. If the pH exceeds 7.4, adjust it with concentrated        HCl.    -   Bring the final volume to 1 liter using a 1000 mL graduated        cylinder. Filter the solution through a 0.22 μm filter unit.    -   Store at 2°-8° C. for up to 6 months.    -   Column Storage Buffer (0.05% w/v NaN₃ in Water)    -   Weigh 50±5 mg of Sodium Azide and add it to a 1000 mL beaker        with a magnetic stir bar.    -   Add 500 mL of HPLC grade water to the beaker and stir until        completely dissolved.    -   Bring the volume to 1 L with water. Filter the solution through        a 0.22 μm filter unit and pour into a 1000 mL HPLC reagent        bottle.    -   Store at room temperature for up to 6 months.

Preparation of High Molecular Weight Species and System SuitabilityStandard

This will provide for a three-fold dilution of heated to unheatedCTLA4-Ig reference material and a 15%-30% High Molecular Weight Speciesarea percent amount. In a 15 mL Falcon test tube, prepare approximately3 mL of reference material at 10.0±1.0 mg/mL using CTLA4-Ig dilutionbuffer. The initial concentration of CTLA4-Ig is from the COA. Make the10.0±1.0 mg/mL dilution from that value. Transfer 1.0 mL of the dilutedreference material, made in into a 2.0 mL cryovial using a 1 mLpipettor, and heat it in a water bath at 67±2° C. for 45±2 minutes.Transfer the heated reference material made in back into the 15 mL testtube using a 1 mL pipettor. Gently pipet up and down a 1 mL volume ofthe contents of the test tube for a total of 10 times. This is thesystem suitability standard. Storage conditions: Prepare 150 μL aliquotsof the system suitability standard in 2.0 mL cryovials for storage at−80±5° C. for up to 1 year. Obtain the initial concentration ofReference Material, and make the 10.0±1.0 mg/mL dilution from thatvalue. Use a minimum of 100 μl of reference material and an appropriateamount of the dilution buffer to achieve a final concentration of10.0±1.0 mg/mL. Refer to the following equation for dilutions

${{Dilution}\mspace{14mu} {Buffer}\mspace{14mu} {to}\mspace{14mu} {Add}} = {\frac{\left( {{Aliquot}\mspace{14mu} {Volume} \times {Starting}\mspace{14mu} {Concentration}} \right)}{{Target}\mspace{14mu} {Concentration}} - {{Aliquot}\mspace{14mu} {volume}}}$

For CTLA4-Ig Drug Substance make the 10.0±1.0 mg/mL dilution from theprotein concentration using a minimum of a 100 μL aliquot of sample andan appropriate amount of the dilution buffer to achieve a finalconcentration of 10.0±1.0 mg/mL. Refer to the following equation fordilutions:

${\frac{\left( {0.1\mspace{14mu} {mL} \times 50\mspace{14mu} {mg}\text{/}{mL}} \right)}{10\mspace{14mu} {mg}\text{/}{mL}} - {0.1\mspace{14mu} {mL}}} = {0.4\mspace{14mu} {mL}\mspace{14mu} {dilution}\mspace{14mu} {buffer}}$

Once diluted to 10.0±1.0 mg/mL, the samples can be stored at 2° C.-8° C.for up to 24 hours. For CTLA4-Ig Drug Product make the 10.0±1.0 mg/mLdilution from the protein concentration using a minimum of a 200 μLaliquot of sample, and an appropriate amount of the dilution buffer toachieve a final concentration of 10.0±1.0 mg/mL. Refer to the followingequation for dilutions:

${\frac{\left( {0.2\mspace{14mu} {mL} \times 25\mspace{14mu} {mg}\text{/}{mL}} \right)}{10\mspace{14mu} {{mg}/{mL}}} - {0.2\mspace{14mu} {mL}}} = {0.3\mspace{14mu} {mL}\mspace{14mu} {dilution}\mspace{14mu} {buffer}}$

Place the Mobile Phase in one solvent reservoir and HPLC grade water inanother. Sonicate and vacuum degas prior to run. Turn on detector andallow 15 minutes to warm up prior to the run. Before a new or currentcolumn is used for analysis, flush the column with HPLC grade water forat least 20 minutes followed by mobile phase buffer equilibration for atleast 20 minutes. Use a flow rate of 1.0 mL/min. Thaw a 150 μL aliquotof system suitability standard, add it to an autosampler vial and placethe vial in the autosampler. Inject 20 μL of the standard under theconditions.

Determine the percentage of High Molecular Weight Species eluting atapproximately 7.5 minutes according to the following formula: (In theformula below, Monomer actually refers to dimer.)

${{Area}\mspace{14mu} \% \mspace{14mu} {High}\mspace{14mu} {Molecular}\mspace{14mu} {Weight}\mspace{14mu} {Species}} = {\frac{(A)}{(A) + (B)} \times 100}$

-   -   Where:    -   A=Peak area of High Molecular Weight Species    -   B=Peak area of Monomer

The Area Percent High Molecular Weight Species should not be less than15%. If it is less than 15%, add additional concentrated High MolecularWeight Species to the system suitability standard above. Resolution (R)Determination and Retention Time Evaluation. Inject 20 μL of systemsuitability standard. Calculate the resolution between the HighMolecular Weight Species (retention time approximately 7.5 minutes) andthe Monomer peak (retention time approximately 8.7 minutes) using thefollowing equation: (In the formula below, “monomer” actually refers todimer.)

${{Resolution}\mspace{14mu} (R)} = \frac{2\left( {t_{2} - t_{1}} \right)}{W_{2} + W_{1}}$

-   -   Where:    -   t₁=Retention time of the high molecular weight species    -   t₂=Retention of time of Monomer    -   W₁=Peak width of high molecular weight species    -   W₂=Peak width of Monomer

Peak width is measured in minutes at the base of the peak afterextrapolating the relatively straight sides of the peak to the baseline.Retention time and peak width are measured in the same units. In oneembodiment, (R) must be ≥1.2 and the retention time for he peak shouldbe 8.7±1.0 minutes.

Number of Theoretical Plates Determination (N)

From the system suitability standard chromatogram, determine theefficiency of the column by calculating the number of theoretical plates(N) according to the following equation:

$N = {16\left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   t=is the retention time of Monomer (in minutes)    -   w=is the width (in minutes) at baseline of the Monomer obtained        by extrapolating the sides of the peak to the base line.

A total of six (6) injections of CTLA4-Ig material will be made. Aliquot200 μL of 10 mg/mL reference material into an autosampler vial, andplace the vial in the autosampler and inject six times. Process thechromatograms, and calculate the dimer peak area for each chromatograph.Sum the dimer peak areas from the six chromatographs and calculate theaverage, standard deviation and % RSD according to the followingequations:

$\frac{X_{1} + X_{2} + {X_{3}\mspace{14mu} \ldots \mspace{14mu} X_{n}}}{n_{x}}$

-   -   Where:    -   X_(1,2,3) . . . =a specific value in a set of data    -   n_(x)=a set of values (x)    -   5.4.4.2 Calculate the standard deviation as follows:

$\sqrt{\frac{{n{\sum x^{2}}} - \left( {\sum x} \right)^{2}}{n\left( {n - 1} \right)}}$

-   -   Where:    -   n=number of values (x) in a set of data    -   x=one value in a set of data    -   5.4.4.3 Calculate the % Relative Standard Deviation (RSD) as        follows:

${\% \mspace{11mu} {RSD}} = {\frac{{Standard}\mspace{14mu} {Deviation}}{Mean} \times 100}$

Example of an Injection Sequence:

SAMPLE INJECTION Dilution Buffer Blank 1 Injection Systeme Suitability 1Injection Reference Material 6 Injections Sample #1 2 Injections Sample#2 2 Injections Sample #3 2 Injections Reference Material 1 InjectionDilution Buffer Blank 1 Injection

Integration of Peaks

Integrate all peak areas in the chromatogram from 5.5 to 11.8 minutes.Enlarge baseline of chromatogram to ensure total area of all LMW and HMWspecies are included in the integration. Disregard peaks in the samplethat correspond to peaks in the control. The inclusion volume peak(11.8-13.5 minutes) and peaks after are not considered in thiscalculation. However, if the area at the inclusion volume is 0.1% orgreater of the total area, it should be noted. The dimer peak elutes at8.7±1.0 minutes, the high molecular weight species peak elutes at7.5±1.0 minutes, and the low molecular weight species (e.g., monomer, ifpresent) will elute after the dimer peak. Any peak other than the highmolecular weight species, dimer or low molecular weight species that has280 nm absorbance in excess of 0.1 area % of the total peak area shouldbe noted. Calculate the area percentages as follows: (The reference to“Abatacept Monomer” in this example refers to CTLA4-Ig dimer.)

$\begin{matrix}{{{Area}\mspace{14mu} \% \mspace{14mu} {High}\mspace{14mu} {Molecular}\mspace{14mu} {Weight}\mspace{14mu} {Species}} = {\frac{(B)}{(A) + (B) + (C)} \times 100}} & {7.2{.1}} \\{{{{Area}\mspace{14mu} \% \mspace{14mu} {Low}\mspace{14mu} {Molecular}\mspace{14mu} {Weight}\mspace{14mu} {Species}} = {\frac{(C)}{(A) + (B) + (C)} \times 100}}{{{Area}\mspace{14mu} \% \mspace{14mu} {Monomer}} = {100 - \left( {{{Area}\mspace{14mu} \% \mspace{14mu} {HMW}} + {{Area}\mspace{14mu} \% \mspace{14mu} {LMW}}} \right)}}} & {7.2{.2}}\end{matrix}$

-   -   Where:    -   A=Abatacept Monomer peak area    -   B=Total area of all peaks with retention times less than        Abatacept Monomer    -   C=Total area of all peaks with retention times greater than        Abatacept Monomer peak (excluding inclusion volume).

Samples were separated by size exclusion chromatography using a Water'sALLIANCE® 2695 (Milford, Mass.) on two 7.8×300 mm TSK Gel G3000SWXL™columns (Tosoh Biosep, Montgomery, Pa.) placed in series employing a6.0×40 mm guard column. 25 microliters of each purified sample wasinjected and separated under isocratic conditions using 0.1M Na₂HPO₄,0.1M Na₂SO4, pH 6.8 at 1.0 mL/min. Samples were detected at 280 nm usinga 996 PDA (photodiode array) detector (Waters, Milford, Mass.) andanalyzed using MILLENNIUM 4.0™ chromatography software (Waters, Milford,Mass.).

The overlaid chromatograms of native and induced single chain materialfrom the denaturing analytical tandem size exclusion chromatography showan average retention time (6 replicates) of 27.96±0.02 and 27.99±0.02,respectively. The average peak area for native single chain was found tobe 495525.0±9589.6 and for induced single chain to be 463311.8±7997.2(Table 13). The overlaid MALDI-TOF spectra of native and induced singlechain material have peaks that are quite broad with a baseline width ofabout 15000 mass units. This is expected due to the heterogeneity of theglycosylation in both samples. The apex point of the single chain peakin each mass spectrum was used to calculate the average mass. Theaverage masses for native and induced CTLA4-Ig single chain are45694.426±297.735 and 45333.086±264.778 mass units respectively based onthe analysis of six replicates (Table 14). The native single chain massis expected to be higher than that of induced single chain, because onthe native single chain there is an extra cysteine (residue mass 103dalton) on Cys¹⁴⁶ of SEQ ID NO:2, whereas the induced single chain is aresult of selectively reducing a single interchain disulfide bridgefollowed by alkylation that adds an acetyl group (mass 58 dalton).

These data show that the native and induced materials produce equivalentresults by the tandem denaturing SEC chromatography and MALDI-TOFanalyses. These results demonstrate comparability between the native andinduced CTLA4-Ig single chain materials.

TABLE 13 HPLC SEC Data of Native and Induced Single Chain Induced NativeRetention Time Peak Area Retention Time Peak Area Replicates (min)(|xV * sec) (min) (|xV * sec) 1 27.985 470199 27.968 505576 2 27.979464424 27.941 499800 3 27.977 469509 27.934 505708 4 27.998 46908127.954 482472 5 28.027 453103 27.991 490434 6 28.016 453555 27.985489160 Average 27.997 463311.8 27.962 495525.0 Stdev 0.021 7997.2 0.0239589.6 % RSD 0.074 1.726 0.083 1.935

TABLE 14 MALDI-TOF Mass Spectrometry Data of Native and Induced SingleChain Replicates Induced SC mass Native SC mass 1 45397.896 45597.475 245432.199 45621.300 3 45256.929 45543.839 4 45433.849 45555.893 545381.812 45634.620 6 45348.376 45340.712 Average 45375.177 45548.973Stdev 66.265 108.043 % RSD 0.15 0.24

The presence of cysteines within a polypepetide chain permits formationof disulphide bonds, which can be intramolecular or intermolecularleading to formation of dimer or multimer protein complexes. CTLA4-Igexists as a dimer (wherein the dimer is made up of two monomers havingany one of the following sequences: (i) 26-383 of SEQ ID NO:2, (ii)26-382 of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, (iv) 26-382 of SEQID NO:2), (v) 25-382 of SEQ ID NO:2, and (vi) 25-383 of SEQ ID NO:2 heldtogether by one disulfide bond between C¹⁴⁶ on each chain. The reductionof this disulfide bond can result in the formation of two equivalentprotein chains held together by non-covalent electrostatic forces. Whensubjected to denaturing conditions that overwhelm or outweigh theattracting electrostatic forces, such CTLA4-Ig can completely dissociateresulting in two identical protein structures of approximately 46 kDa.The resulting structure is referred to as single chain or monomer. Thepresence of the single chain can be monitored by tandem column sizeexclusion chromatography run under denaturing conditions.

A subpopulation of CTLA4-Ig molecules contains a modification on Cys¹⁴⁶:the disulfide linkage present in the majority population is changed to afree cysteine amino acid (referred to as a cysteinylation). CTLA4-Igdimer is predominantly a protein with a molecular weight ofapproximately 92,000. It is comprised of two glycosylated polypeptidechains which are held together by one inter-chain disulfide bond atCys¹⁴⁶ and non-covalent interactions (also referred to as CTLA4-Ig“dimer”). The purified protein exists as a heterogeneous population andcontains modifications such as glycosylations and variations at theN-and C-termini.

A distinct population of CTLA4-Ig molecules exists, which lacks theinterchain disulfide bond linkage. This non-covalently linked populationexists within the frontal dimer peak generated by size exclusionchromatography. The frontal dimer was found to lack the interchaindisulfide bond. The CTLA4-Ig species which lack the interchaindisulphide link are modified by cysteinylation at Cys¹⁴⁶, whichmodification occurs on >99% of the single chain species based on theESI-MS intact data. Cys¹⁴⁶ cysteinylation and the enrichment of O-linkedcarbohydrates are the two major modifications on the CTLA4-Ig singlechain species. The frontal dimer is subjected to denaturing sizeexclusion chromatography resulting in isolation of the CTLA4-Ig singlechain peak. The purified frontal monomer material was compared toCTLA4-Ig with and without solid phase extraction (SPE) and analyzed onMALDI. The purified frontal monomer contains two dominant species: amajor species at either 47005 u for SPE-treated or 46897 u fornon-treated; a minor species at either 95070 u for SPE-treated or 96172u for non-treated. The CTLA4-Ig material also contains two dominantspecies: a major species at either 91518 u for SPE-treated or 91143 ufor non-treated; a minor species at either 45660 u for SPE-treated or46014 u for non-treated.

1 example 2 example Size Homogeneity ≥95.5 Area % ≥95.5 Area % (HPLC)Dimer ≥97.0% Area % ≥97.0% Area % Size Homogeneity ≤3.0 Area %. ≤3.0Area %. (HPLC) High MW ≤2.0 Area %. ≤2.0 Area %. Species SizeHomogeneity ≤0.5 Area % ≤0.5 Area % (HPLC) Low MW Species (monomer)

The CTLA4-Ig composition, in one embodiment, have the followingcharacteristics as to Size Homogeneity (HPLC) analysis of dimer, High MWSpecies, and Low MW Species (monomer, single chain):

-   ≥97.0% Dimer-   ≤2.0% HMW species-   ≤0.5% LMW species (e.g., monomer, single chain)-   In another embodiment, the CTLA4-Ig composition has the following    characteristic amounts of each species:-   ≥95.5% dimer-   ≤3.0% HMW species-   ≤0.5% LMW species (e.g., monomer, single chain)

Summary of SE-HPLC Analysis of Process CD-CHO1 Batches Process CD-CHO1 n= 109 dimer HMW Average (%) 99.4 0.6 % CV 0.3% 45.7% Minimum (%) 98.40.2 Maximum (%) 99.8 1.6 95% Tolerance Interval ≥98.7 ≤1.3

The percent monomer ranged from 98.4 to 99.8% with an average value of99.4% for drug substance manufactured using Process CD-CHO1. Thepercentage HMW species varied from 0.2 to 1.6%. The average value was0.6% with a CV of 45.7%. The 95% tolerance interval was ≥98.7% for thedimer and ≤1.3% for the HMW species. The percent HMW species of thebatches varied from a minimum value of 0.4% to a maximum value of 2.1%.The average percent HMW species was 0.8% with a % CV of 40%. In allcases, the LMW or monomer species were below the detection limit(DL=0.1%). The 95% tolerance interval (to provide coverage for 99 area %of the population) for CTLA4-Ig manufactured by Process CD-CHO1 were≤1.3 and ≤1.8% respectively for the HMW species. The 95% toleranceintervals for the dimer in drug substance from Process CD-CHO1 were98.7% and 96.5% respectively.

Summary of combined data for CTLA4-Ig dimer (n = 143) HMW (n = 141)^(a)Average (%) 99.3 0.6 % CV 0.5 46.9 Minimum (%) 94.8 0.2 Maximum (%) 99.82.1 95% Tolerance Interval ≥97.3 ≤1.8 % CV (between-site) 0.3% 26.4% %CV (within-site) 0.5% 44.2% % CV (total-site) 0.6% 51.4%

The above Table shows that the percent HMW species ranged from 0.2 to2.1% with an average of 0.6%. The dimer ranged from 94.8% to 99.8% withan average value of 99.3% and a precision of 0.3%. The variationbetween-site, within-site and total-site variation for the dimer waswithin 0.3 to 0.5%. The between site variation for the HMW was 26.4%.The within-site and total-site variation was for the percent HMW species44.2 and 51.4% respectively. The 95% tolerance interval for the dimer(97.3%) and the HMW species (1.8%) were within the specification.

Example 11 Vector Construction

Construction of the pcSD Expression Vector: The expression vector, pcSDwas constructed from the commercially available pcDNA3 vector(Invitrogen, Carlsbad, Calif.) as shown in FIG. 27. The neomycinresistance gene cassette was removed from plasmid pcDNA3 by digestionwith restriction endonuclease Nae I. The restriction endonuclease Nae Icreates blunt ends. The DNA fragments were separated by agarose gelelectrophoresis and the 3.821 kb pcDNA3 vector backbone was purifiedfrom the gel. The DNA fragment containing the gene coding for mousedihydrofolate reductase (dhfr) and an SV40 promoter was isolated fromplasmid pSV2-dhfr by digestion of the plasmid with restrictionendonucleases Pvu II and BamH I. The 1.93 kb fragment corresponding tothe dhfr gene cassette was separated and purified by agarose gelelectrophoresis. The 3-prime recessed ends generated by BamH I digestionwere filled in using the Klenow fragment of DNA polymerase I to generateblunt ends. This isolated fragment was ligated to the blunt-ended 3.8 kbpcDNA3 vector backbone to create the expression vector pcSD. Thisexpression vector has the following features: a cytomegalovirus (CMV)promoter followed by a multiple cloning site, a bovine growth hormone(BGH) polyadenylation signal and transcriptional termination sequence, amouse dhfr cDNA sequence for selection and amplification, and anampicillin resistance gene and pUC origin of replication for selectionand maintenance in Escherichia coll.

Construction of the pcSDhuCTLA4-Ig Expression Vector: A 1.2 kilobase(kb) DNA fragment containing a sequence encoding a CTLA4-Ig protein wasisolated from plasmid pCDM8-CTLA4-Ig by digestion with the restrictionenzymes Hind III and Xba I. The 1.2-kb Hind III/Xba I fragment wasligated into vector piLN previously digested with the restrictionenzymes Hind III and Xba I. The resulting plasmid construct, designatedpiLN-huCTLA4-Ig, is shown in FIG. 20. The piLN-huCTLA4-Ig plasmid wasused as the source of the CTLA4-Ig coding sequence used in theconstruction of the final expression vector pcSDhuCTLA4-Ig.

The final vector for expression of the CTLA4-Ig gene was constructed asshown in FIG. 28. A 1.2 kb DNA fragment containing the CTLA4-Ig gene wasisolated from plasmid piLN-huCTLA4-Ig by a two step restriction digestprocedure. Plasmid piLN-huCTLA4-Ig was first digested with restrictionenzyme Hind III. The resulting 3-prime recessed ends were filled in bytreatment with the Klenow fragment of DNA polymerase I. The plasmid wasthen digested with the restriction enzyme Xba I to release the 1.2 kbfragment containing the CTLA4-Ig gene. This fragment was purified andligated to the EcoR V and Xba I fragment isolated from the restrictiondigestion of pcSD. The EcoR V and Xba I restriction sites are located inthe multiple cloning site of pcSD between the CMV promoter and acassette containing the bovine growth hormone polyadenylation signal andtranscriptional termination sequence. This placed the CTLA4-Ig genefragment under the control of the CMV promoter. This plasmid isdesignated pcSDhuCTLA4-Ig, and comprises SEQ ID NO:1.

Example 12 Transfection of CTLA4-Ig Expression Vector to Obtain StableCell Lines

This Example and Example 13 describe a newly transfected population ofcells from which individual clones were selected and expanded, and thusthe expanded clones are different than the cells deposited with the ATCCas Accession No. CRL-10762. The previous CHO cell line harboring anexpression vector containing DNA encoding the amino acid sequencecorresponding to CTLA4-Ig (DNA having ATCC Accession Number 68629) isdescribed in U.S. Pat. No. 5,434,131. Briefly, an expression plasmid(for example, pCDM8) containing cDNA that was deposited under ATCCAccession No. 68629, was transfected by lipofection using standardprocedures into dhfr negative-CHO cells to obtain cell lines that stablyexpress CTLA4-Ig. Screening B7 positive CHO cell lines for B7 bindingactivity in the medium using immunostaining resulted in a stabletransfectant that expressed CTLA4-Ig. This heterogenous population oftransfected cells was designated Chinese Hamster Ovary Cell Line,CTLA4-Ig-24 and was deposited with the ATCC under the Budapest Treaty onMay 31, 1991 having ATCC Accession Number CRL-10762.

The Chinese hamster ovary cell line, DG44, contains a deletion of thegene coding for the enzyme dihydrofolate reductase. The expressionplasmid pcSDhuCTLA4-Ig contains a copy of the dihydrofolate reductasegene (dhfr). Insertion of plasmid pcSDhuCTLA4-Ig into the DG44 genomeresults in functional complementation of the dhfr deletion. Thisfunctional complementation can be used for selection of transfectants inthe presence of methotrexate (MTX) and amplification of dhfr andadjacent genes.

The human CTLA4-Ig-secreting cell line 1D5 was constructed bytransfection of cell line DG44 with the pcSDhuCTLA4-Ig expressionplasmid as shown in FIG. 28. The plasmid DNA was introduced into DG44cells by electroporation using standard procedures known in the art.Transfectants were selected using a minimal essential medium (MEM; JRHBiosciences, Inc., Kansas) supplemented with 5% (v/v) dialyzed FetalBovine Serum. Culture supernatants from the transfectants were screenedfor human IgG production using a sandwich ELISA method. An Fc-specificgoat anti-human IgG was used as the capture antibody. Goat anti-humanIgG antibody conjugated to horseradish peroxidase was used to detecthuman IgG. Transfectants expressing higher levels of the human CTLA4-Iggene were selected for further amplification.

Gene amplification of the selected transfectants was accomplished byaddition of MTX to the culture medium at a final concentration of 100nM. MTX is a folic acid analogue that acts as a competitive inhibitor ofdihydrofolate reductase. Addition of MTX to the medium allowed selectionof transfectants containing multiple copies of the dhfr gene andelevated levels of dihydrofolate reductase. Transfectants alsocontaining multiple copies of the adjacent CTLA4-Ig gene were identifiedusing the human IgG specific ELISA method. A CTLA4-Ig-producing clone,designated 1D5, was selected for further development, in part describedin Example 13.

Example 13 Subcloning of Stably Transfected Cells

Cell line 1D5 was subjected to soft agar cloning. Subclones derived fromthe soft agar cloning were analyzed for human IgG production using theELISA method. Selected subclones were evaluated for CTLA4-Ig productionand growth properties. The lead subclone, designated 1D5-100A1, wasselected.

The 1D5-100A1 cell line was adapted from DE medium (JRH Biosciences,Inc., Kansas, which contains animal-sourced raw materials, to achemically defined medium designated CD-CHO (Table 15). CD-CHO medium isa proprietary, animal component-free medium manufactured by InvitrogenCorporation, Carlsbad, Calif.

TABLE 15 Composition of Process Y Medium Component Concentration CD-CHO25x Acid Solubles I 40.0 mL/L CD-CHO 25x Acid Solubles II 40.0 mL/LCD-CHO 25x Salts I 40.0 mL/L CD-CHO 25x Salts II 40.0 mL/L L-Glutamine0.585 g/L r-human Insulin (10 mg/mL solution) 0.1 mL/L Methotrexate (20mM solution) 5 μL/L Sodium Bicarbonate 2.22 g/L Water As required 1N HClSolution 0-5 mL/L to adjust pH 10N NaOH Solution 0-10 mL/L to adjust pH

Cell line 1D5-100A1 was cultured and passaged in DE medium according tostandard tissue culture protocol. The cells were then transferred to amedium composed of 50% DE medium and 50% CD-CHO medium. After severalpassages in this medium, the cells were transferred to T-flaskscontaining 100% CD-CHO medium. The cells were grown in 100% CD-CHOmedium for several passages. The adapted cells were then subjected tocloning by limiting dilution.

Cells from the CD-CHO-adapted 1D5-100A1 cell line were cloned bylimiting dilution using serum-free media. The 1D5-100A1 cells wereseeded at a target of 1 cell/well into 96-well microtiter platescontaining supplemented MCDB medium. MCDB is a chemically defined mediumformulation distributed by Sigma-Aldrich, St. Louis, Mo. The MCDB mediumwas supplemented with 4 mM glutamine, 500 μg/mL recombinant humaninsulin, 100 nM MTX and 10% conditioned medium. The conditioned mediumwas a filter-sterilized supernatant from a culture of the CD-CHO adapted1D5-100A1 cell line grown in MCDB medium.

Wells containing a single colony were identified and the clonesevaluated for CTLA4-Ig production using the ELISA method. Selectedclones were expanded from 96-well microtiter plates to 6-well cellculture plates. The cultures were further expanded into 25 cm² T-flasksand then roller bottles.

The roller bottle cultures were evaluated for CTLA4-Ig titer, CTLA4-Igsialic acid content, and growth. Three clones were selected for furtherevaluation in bioreactors and further characterization. A frozen vialresearch stock of clonal cell line 1D5-100A1.17 stored at −80° C. wasused to generate a cell bank.

Example 14 Production of CTLA4-Ig in Bioreactors via a Fed-Batch Process

Commercial Scale Culturing of Suspension Mammalian Cells ExpressingCTLA4-Ig: This Example describes the production of CTLA4-Ig moleculescomprising SEQ ID NO:2 monomers, from suspension cultured dhfr-negativeCHO cells. The methods described in this Example can be adapted andextended for the production of other recombinant proteins, including butnot limited to, secreted proteins such as cytokines and other hormones,secreted proteins that are members of the Ig superfamily or comprise aportion of an Ig superfamily protein, and generally any proteinexpressed in CHO cells.

The culture flasks (for example, T-175 and Erlenmyer flasks), rollerbottles, and cell bags were used for the inoculum expansion steps of theCTLA4-Ig culturing process to serially propagate cells from a frozenvial to provide a sufficient number of viable cells to inoculate a20,000-L bioreactor.

A single vial of cells is removed from the vapor phase of a liquidnitrogen storage freezer and thawed in a water bath at 37° C. The entirecontents of the vial are aseptically transferred into a sterile 15-mLconical centrifuge tube. CD-CHO medium is added to bring the finalvolume to 10 mL. The cell suspension is centrifuged, the supernatantdiscarded and the cell pellet resuspended in 10 mL of CD-CHO cellculture medium. The resuspended cells are transferred to a T-175 flaskcontaining 10 mL of CD-CHO medium. The viable cell density and thepercent viability of the culture in the T-175 flask is determined. Acriterion for the percent viability at this step of 84% was established.CD-CHO medium is added to the T-175 flask to achieve a target viablecell density of 1.7-2.6×10⁵ cells/mL.

The T-175 flask is incubated at 37° C. in an atmosphere of 6% carbondioxide for a maximum of four days to achieve a target final cell numberof ≥6×10⁶ viable cells. Following the T-175 flask step, the culture isexpanded using a series of shaker flasks, 1-L, and 2-L Roller Bottles.At each passage, the cells are seeded at a target density of 2.0×10⁵viable cells/mL, wherein cultures targeted at having a final culturecell viability ≥80%. The cultures are incubated in CD-CHO medium at 37°C. in an atmosphere of 6% carbon dioxide for a maximum of four days.

Expansion of the culture occurs in a series of cell bags (20-L, 100-L,and 200-L) in order to further inoculate a 1000-L bioreactor. Cellculture material from the 2-L roller bottles inoculum expansion step ispooled to inoculate a 20-L cell bag at a target seeding density of2.0×10⁵ viable cells/mL. A condition for the final viable cell densityat the 2-L roller bottle inoculum expansion step of 1.0 to 2.0×10⁶cells/mL and a minimum percent cell viability of 80% were established.Upon inoculation, the 20-L cell bag culture is incubated in CD-CHOmedium at 37° C. in an atmosphere of 6% carbon dioxide for a maximum offour days. For each subsequent passage (100-L and 200-L cell bags), thecells are seeded at a target density of 2.0×10⁵ viable cells/mL, whereincultures targeted at having a final culture cell viability ≥80%. Thecultures are incubated in CD-CHO medium at 37° C. in an atmosphere of 6%carbon dioxide for a maximum of four days. Exemplary values for thefinal viable cell density at the 20-L, 100-L, and 200-L cell baginoculum expansion step of 1.0 to 2.0×10⁶ cells/mL and a minimum percentcell viability of ≥80% were established. These exemplary values ensurethat a sufficient number of viable cells is used to inoculate the 1000-Lbioreactor.

The objective of the 1000-L and 4000-L seed bioreactor inoculumexpansion steps of the CTLA4-Ig process is to provide a sufficientnumber of viable cells to inoculate the 20,000-L production bioreactor.

The seed bioreactors are operated in batch mode using CD-CHO cellculture medium. Temperature, pH, dissolved oxygen, pressure, agitationand gas flow rates for air, oxygen, and carbon dioxide are controlled bya distributed control system (DCS) and provide conditions for optimalgrowth of the culture in the seed bioreactors. The seed bioreactors areoperated at 37° C. Culture samples are removed from the seed bioreactorsfor the determination of viable cell density, percent viability, andmetabolite concentrations.

The 1000-L seed bioreactor is inoculated with inoculum from the 200-Lcell bag expansion step to a target initial viable cell density of 1.0to 3.0×10⁵ viable cells/mL. The culture is incubated in CD-CHO medium at37° C. for a maximum of 5 days. Exemplary values for the final viablecell density at the 1000-L seed bioreactor inoculum expansion step is1.0 to 2.0×10⁶ cells/mL and a minimum percent cell viability is ≥80%.

The 4000-L seed bioreactor is inoculated with inoculum from the 1000-Lseed bioreactor expansion step to a target initial viable cell densityof 1.0 to 3.0×10⁵ viable cells/mL. The culture is incubated in CD-CHOmedium at 37° C. for a maximum of 6 days. Exemplary values for the finalviable cell density at the 4000-L seed bioreactor inoculum expansionstep is 1.0 to 2.0×10⁶ cells/mL and a minimum percent cell viability is≥80%. These exemplary values ensure that a sufficient number of viablecells is used to inoculate the 20,000-L production bioreactor.

The 20,000-L seed bioreactor is inoculated with inoculum from the 4000-Lseed bioreactor expansion step to a target initial viable cell densityof 1.0 to 1.8×10⁵ viable cells/mL. The culture is incubated in CD-CHOmedium at 37° C. for a maximum of 6 days. Exemplary values for the finalviable cell density at the 20,000-L seed bioreactor inoculum expansionstep is 1.0 to 2.0×10⁶ cells/mL and a minimum percent cell viability is≥80%. These exemplary values ensure that a sufficient number of viablecells is used prior to initiating the production phase in the 20,000-Lproduction bioreactor.

Commercial Scale Production of CTLA4-Ig: The production phase of thisinvention occurring in a 20,000-L production bioreactor produces bothhigh quantity and high quality CTLA4-Ig protein, which involves cultureruns having a two-step temperature shift. The 20,000 L culture that isincubated in CD-CHO medium at 37° C. for a maximum of 6 days (asdescribed above) is subjected to a temperature shift (T-shift) from 37°C. to 34° C. on day 6 (the end of logarithmic growth phase). Twelvehours after the 37° C. to 34° C. temperature-shift, CD-CHO medium issupplemented with a modified eRDF feed medium (Invitrogen Corp.,Carlsbad, Calif.; Tables 16, 17), and this feed is provided daily to theproduction reactor as a bolus (1% w/w).

TABLE 16 Composition of eRDF Feed Medium Component Concentration eRDF-1Medium (Invitrogen Corp.) 16.47 g/kg Dextrose 30.29 g/kg D-Galactose12.38 g/kg L-Glutamine 4.02 g/kg r-human Insulin (10 mg/mL solution)0.98 mL/kg TC Yeastolate 4.90 g/kg Water As required 1N HCl Solution 0-5mL/kg to adjust pH 10N NaOH Solution 0-2 mL/kg to adjust pH

TABLE 17 Composition of eRDF-1 Medium Component Concentration (mg/L)Cupric Sulfate 5 H₂O 0.0008 Ferrous Sulfate 7 H₂O 0.220 MagnesiumSulfate (MgSO₄) 66.20 Zinc Sulfate 7 H₂O 0.230 Sodium Pyruvate 110.0DL-Lipoic Acid Thioctic 0.050 Linoleic Acid 0.021 L-Alanine 6.68L-Arginine 581.44 L-Asparagine 94.59 L-Aspartic Acid 39.93 L-Cystine 2HCl 105.38 L-Glutamic Acid 39.7 Glycine 42.8 L-Histidine HCl—H₂O 75.47L-Isoleucine 157.40 L-Leucine 165.30 L-Lysine HCl 197.26 L-Methionine49.24 L-Phenylalanine 74.30 L-Proline 55.3 L-Hydroxyproline 31.5L-Serine 85.10 L-Threonine 110.8 L-Tryptophan 18.40 L-Tyrosine 2 Na 2H₂O108.10 L-Valine 108.9 Para Amino Benzoic Acid 0.51 Vitamin B12 0.339Biotin 1.00 D-Ca Pantothenate 1.29 Choline Chloride 12.29 Folic Acid1.96 i-Inositol 46.84 Niacinamide 1.47 Pyridoxal HCl 1.00 Pyridoxine HCl0.420 Riboflavin 0.21 Thiamine HCl 1.59 Putrescine 2HCl 0.020

The 20,000 L culture is incubated in CD-CHO medium supplemented dailywith eRDF feed medium at 34° C. for a maximum of 4 days. On day 10, the20,000 L culture is subjected to a second T-shift from 34° C. to 32° C.The 20,000 L production culture in the production bioreactor wasmaintained at 32° C. for a maximum of 8 days. On day 18, a culturesample was analyzed for the following exemplary values: viable celldensity at the 20,000-L seed bioreactor production step is 3.0 to8.0×10⁶ cells/mL; minimum percent cell viability is ≥38%; final sialicacid molar ratio (described elsewhere) is ≥6; and final CTLA4-Ig proteinproduct titer is 0.5 to1.3 g/L. These exemplary values ensure that aprotein product of sufficient quality and quantity is being produced bythe recombinant CHO cell line and that the 20,000-L mammalian cellculture is ready to be harvested.

The culture in the bioreactor during the production phase is given adaily bolus feed using modified eRDF medium (Table 16, 17), as follows:starting 12 hours after the initial temperature shift (37° C. to 34°C.), a minimum of 1% culture volume was added as feeding medium; if theglucose level fell below 3 g/L, a calculated volume is added to bringthe glucose level back to 3 g/L.

The production phase had duration of 18 days at the 20,000 L scale.Samples were taken on a daily basis from the production bioreactor foranalysis. For example, a sample used for cell counting was stained withtrypan blue (Sigma, St. Louis, Mo.). Cell count and cell viabilitydetermination was performed using a hemocytometer to count viablestained cells under the microscope. For analysis of metabolites, anadditional sample aliquot was centrifuged for 20 minutes at 2000 rpm (4°C.) to pellet the cells. The supernatant was analyzed for protein titer,sialic acid, glucose, lactate, glutamine, glutamate, pH, pO₂, pCO₂,ammonia, and LDH, using techniques and protocols conventionallypracticed in the art.

Example 15 Purification of Recombinant CTLA4-Ig

QXL Anion Exchange Chromatography for CTLA4-Ig Purification: The anionexchange chromatography step in the CTLA4-Ig process uses Q SepharoseExtreme Load (QXL) anion exchange chromatography resin. This resin issupplied by GE Healthcare, Waukesha, Wis. (formerly AmershamBiosciences). The QXL chromatography step is to capture and concentratethe CTLA4-Ig dimer from the in-process material from the harvestoperation steps for further downstream processing.

A 1.0-2.0 m inner diameter column is packed with QXL resin to a heightof 17 to 30 cm, representing a volume of about 643 L to 1018 L. Thecolumn is qualified for use by determining the height equivalent to atheoretical plate (HETP) and asymmetry (A_(s)) of the packed column. AHETP of 0.02 to 0.08 cm and an A_(s) of 0.8 to 1.2 are employed forqualification of the QXL column.

The QXL column operation is carried out at ambient temperature. Theclarified cell culture broth is loaded onto an equilibrated QXL column.The QXL chromatography step is performed using a maximum flow rate of99.4 L/min. The column inlet pressure is maintained below 35 psig. Themaximum CTLA4-Ig protein load for the QXL column is 28 grams of CTLA4-Igper liter of resin.

The QXL chromatography column is first sanitized with a 1 N sodiumhydroxide solution. The sanitization is performed using 2 to 4 columnvolumes (CV) of the 1 N sodium hydroxide solution. The sanitization iscomplete when the conductivity of the column effluent equals 169±33mS/cm and the column is held for 60 to 120 minutes.

After the sanitization step, the column is equilibrated with a 75 mMHEPES, 360 mM sodium chloride, pH 8.0 buffer. The equilibration iscomplete when a minimum of 3 CV of equilibration buffer have been passedthrough the column and the pH of the effluent is 8.0±0.2 and theconductivity of the effluent is 13.4±1.0 mS/cm.

The in-process material from the harvest operation step is loaded ontothe QXL column. The column is washed with a minimum of 10 CV of washbuffer (75 mM HEPES, 360 mM NaCl, pH 8.0), and the absorbance at 280 nm(A₂₈₀) of the column effluent is measured at the end of the wash step.CTLA4-Ig is then eluted from the column with a 25 mM HEPES, 325 mM NaClor 850 mM NaCl, pH 7.0 buffer. The eluate is diverted into a collectionvessel when the A₂₈₀ increases to ≥0.02 absorbance units (AU) above theAU value at the end of the wash step. The eluate is collected until theA₂₈₀ of the trailing edge of the elution peak decreases to a value of≤1.0 AU.

A CTLA4-Ig dimer product with a molar ratio of moles sialic acid tomoles CTLA4-Ig protein that is ≥8 is collected, and a pool of CTLA4-Ighigh molecular weight material is present at ≤25.7%. The CTLA4-Ig highmolecular weight material, which includes tetramers, can then be furtherpurified for use as a separate substance for the methods of treatmentdescribed herein.

Phenyl Sepharose FF HIC for CTLA4-Ig Purification: The hydrophobicinteraction chromatography (HIC) step uses Phenyl Sepharose Fast Flowresin (GE Healthcare, Waukesha, Wis. (formerly Amersham Biosciences)).The HIC step reduces the level of CTLA4-Ig high molecular weightmaterial present in the QXL product pool. The CTLA4-Ig dimer does notbind to the HIC resin under the loading conditions used for the HICstep.

A 1.0 to 2.0 m inner diameter column is packed with Phenyl SepharoseFast Flow resin to a height of 18 to 22 cm, representing a volume ofabout 680 to 852 L. The column is qualified for use by determining theHETP and A_(s) of the packed column. A HETP of 0.02 to 0.08 cm and anA_(s) of 0.8 to 1.2 are employed for qualification of the HIC column.

The HIC column operation is carried out at ambient temperature. Theeluate pool from the QXL column step is loaded without further treatmentonto the equilibrated HIC column. The HIC step is operated at a maximumflow rate of 65.4 L/min and at a operating pressure of 13 psig. Themaximum CTLA4-Ig protein load applied to the HIC column is 10.0 g ofCTLA4-Ig protein per liter of resin. Multiple cycles of the HIC step canbe employed based on the amount of CTLA4-Ig protein present in the QXLeluate pool.

The HIC column is first sanitized with a 1 N sodium hydroxide solution.The sanitization is complete when 2 to 4 CV of the 1 N sodium hydroxidesolution have been passed through the column. The column is then heldfor 60 to 120 minutes to ensure sanitization.

After the sanitization step, the column is equilibrated with a 75 mMHEPES, 2.55 M sodium chloride, pH 7.0 buffer. The equilibration iscomplete when a minimum of 3 CV of equilibration buffer have been passedthrough the column and the pH of the effluent is 7.0±0.3 and theconductivity is 71.5 to 75.5 mS/cm.

The eluate from the QXL step is applied to the equilibrated HIC column.The column is then washed with the chase equilibration buffer until theA₂₈₀ of the effluent decreases to between 0.8 and 1.0 AU. The CTLA4-Igprotein-containing effluent from each cycle of the HIC step is filteredthrough a 0.2 μm cellulose acetate filter into a common stainless steelcollection vessel. This HIC product pool is held in the collectionvessel at 2° to 8° C. The maximum hold time in the collection vessel is3 days.

A CTLA4-Ig dimer product with a molar ratio of moles sialic acid tomoles CTLA4-Ig protein that is ≥8 is collected, and a pool of CTLA4-Ighigh molecular weight material is present at ≤2.5%.

Recombinant Protein A Affinity Chromatography for CTLA4-Ig Purification:The recombinant Protein A Sepharose Fast Flow affinity resin (rPA) usedin the downstream CTLA4-Ig production process is obtained from GEHealthcare (Waukesha, Wis. (formerly Amersham Biosciences)). The rPAcolumn chromatography step further purifies the CTLA4-Ig protein. Thisstep removes DNA and host cell proteins including monocyte chemotacticprotein 1 (MCP-1).

An 80 to 140 cm inner diameter column is packed with rPA resin to aheight of 18 to 25 cm, representing a volume of about 339 to 372 L. Thecolumn is qualified for use by determining HETP and A_(s) of the packedcolumn. A HETP of 0.02 to 0.08 cm and an A_(s) of 0.8 to 1.2 areemployed for qualification of the column. The maximum number of uses forthe rPA resin established in a resin lifetime study is 60.

The rPA column operation is carried out at ambient temperature. Theviral inactivation product pool is loaded onto the equilibrated rPAcolumn. The rPA step is operated at a maximum flow rate of 26.7 L/minand an operating pressure of ≤13 psig. The maximum CTLA4-Ig protein loadapplied to the rPA column is 25 g of CTLA4-Ig protein per liter ofresin.

The rPA column is equilibrated with a 25 mM Tris, 250 mM NaCl, pH 8.0buffer. Equilibration is complete when a minimum of 3 CV ofequilibration buffer have been passed through the column and the pH andconductivity values of the effluent are between 7.8 to 8.2 and 23.0 to27.0 mS/cm, respectively.

The viral inactivation step product pool is applied to the equilibratedrPA column. The rPA chromatography step includes two wash steps. Thefirst wash step is performed using a minimum of 5 CV of a 25 mM Tris,250 mM NaCl, 0.5% Triton X-100, pH 8.0 buffer to remove weakly boundmaterial from the rPA column. The second wash step is performed using a25 mM Tris, 250 mM NaCl, pH 8.0 buffer. The second wash step uses aminimum of 5 CV to remove the residual Triton X-100 from the rPA column.

The CTLA4-Ig protein is eluted from the rPA chromatography column with a100 mM glycine, pH 3.5 buffer. The eluate is diverted into a collectionvessel when the A₂₈₀ increases to ≥0.2 AU above the baseline. The columneffluent is filtered through a 0.2 μm cellulose acetate filter into acollection vessel equipped with an agitator. The eluate is collecteduntil the A₂₈₀ of the trailing edge of the elution peak decreases to avalue of ≤0.2 AU. The pH of the eluate pool is adjusted to pH 7.5±0.2with a 2 M HEPES, pH 8.0 buffer. The rPA chromatography step productpool is held at 2° to 8° C. for a maximum of 3 days.

A CTLA4-Ig dimer product with a molar ratio of moles sialic acid tomoles CTLA4-Ig protein that is ≥8 is collected; a pool of CTLA4-Ig highmolecular weight material is present at ≤2.5%; and a pool of MCP-1 ≤38ng/mL is present.

QFF Anion Exchange Chromatography for CTLA4-Ig Purification: The anionexchange chromatography step in the downstream CTLA4-Ig productionprocess uses Q Sepharose Fast Flow (QFF) anion exchange chromatographyresin (GE Healthcare (Waukesha, Wis. (formerly Amersham Biosciences).The objective of the QFF chromatography step is to reduce the residualProtein A levels and provide additional reduction of host cell DNA fromthe viral filtration step product pool. The QFF column step is also usedto control the sialic acid to CTLA4-Ig protein molar ratio of the QFFchromatography step product pool and to provide additional control ofin-process CTLA4-Ig HMW material levels. The primary in-process controlpoint for the reduction of CTLA4-Ig HMW material is the HIC step.

A 60 to 140 cm inner diameter column is packed with QFF resin to aheight of 28 to 35 cm, representing a volume of about 536 to 667 L. Thecolumn is qualified for use by determining the HETP and A_(s) of thepacked column. A HETP of 0.02 to 0.08 cm and an A_(s) of 0.8 to 1.2 areemployed for qualification of the column.

The QFF column operation is carried out at ambient temperature. Theviral filtration step product pool is loaded onto the equilibrated QFFcolumn. The QFF step is operated at a maximum flow rate of 38.7 L/minand an operating pressure of ≤35 psig. The maximum CTLA4-Ig protein loadapplied to the QFF column is 25 g of CTLA4-Ig protein per liter ofresin.

The QFF chromatography column is first sanitized with a 1 N sodiumhydroxide solution. The sanitization is performed using 2 to 4 CV of the1 N sodium hydroxide solution. The sanitization is complete when theconductivity of the column effluent equals 136 to 202 mS/cm and thecolumn is held for 60 to 120 minutes.

After the sanitization step, the column is equilibrated with a 25 mMHEPES, 100 mM sodium chloride, pH 8.0 buffer. The equilibration iscomplete when a minimum of 4 CV of equilibration buffer have been passedthrough the column and the pH of the effluent is 7.7 to 8.3 and theconductivity is 10.5 to 12.9 mS/cm.

The viral filtration step product pool contained in bioprocess bags istransferred into a sterile stainless steel collection vessel.

The viral filtration step product pool is applied to the equilibratedQFF column. The QFF chromatography step includes two wash steps. Thefirst wash step is performed using a minimum of 5.0 CV of a 25 mM HEPES,120 mM NaCl, pH 8.0 buffer. The second wash step is performed using aminimum 5.0 CV of a 25 mM HEPES, 130 mM NaCl, pH 8.0 buffer.

The CTLA4-Ig dimer is eluted from the QFF chromatography column using a25 mM HEPES, 200 mM NaCl, pH 8.0 buffer. The eluate collection isinitiated when the A₂₈₀ of the effluent begins to increase. Duringelution, the column effluent is filtered through a 0.2 μm celluloseacetate filter into the stainless steel collection vessel. The eluate iscollected until the absorbance of the trailing edge of the elution peakdecreases to ≤0.2 AU above the baseline. The collection vessel is thencooled to 2° to 8° C. The maximum hold time for the QFF chromatographystep product pool at 2° to 8° C. is 3 days.

A CTLA4-Ig dimer product with a molar ratio of moles sialic acid tomoles CTLA4-Ig protein that is ≥8 is collected; a pool of CTLA4-Ig highmolecular weight material is present at ≤2.5%; a pool of CTLA4-Ig lowmolecular weight material (for example CTLA4-Ig monomer) is present at≤0.5%; and a pool of MCP-1 ≤9.5 ng/mL is present.

The Pall Filtron TFF system is used in the concentration anddiafiltration step of the downstream CTLA4-Ig production process. Theobjective of this step is to concentrate the QFF chromatography stepproduct pool to 45 to 55 g/L and to exchange the elution buffer used inthe QFF chromatography step with the final buffer used for CTLA4-Igcompositions. The concentrated CTLA4-Ig protein product pool istransferred through a 0.2 μm polyvinylidene fluoride filter and into a50-L bioprocess bag.

Example 16 CTLA4-Ig-Molar Ratio Determination of Amino Monosaccharides

This example provides methods to obtain molar ratios of aminomonosaccharides (N-acetyl galactosamine, N-acetyl glucosamine) toprotein in CTLA4-Ig samples.

Instrumentation: Capillary Electrophoresis System Beckman P/ACE MDQ CESystem; Detector Beckman Laser-Induced-Fluorescence (LIF) detectionsystem(coupled with P/ACE MDQ); Uncoated capillary (i.d. 25 μm;o.d. 360μm), 27-31 cm total length to accomodate either P/ACE MDQ or 5510PolyMicro Technologies, Cat. No. TSP025375; Maxi-Mix mixer Thermolyne,(VWR Catalog No. 58810-185)

Reagents:

Hydrolysis Solution (4 N HCl Aqueous Solution)

Add 160 mL of 6 N HCl and 80 mL of HPLC grade water to a 250 mL glassbottle.

Stir to mix well.

Store at 2-8° C. for up to 6 months.

Derivatization Solution I (0.1 M APTS Aqueous Solution)

Add 192 4 of HPLC grade water to 10 mg powder of APTS in a glass vial.

Vortex the vial 5-10 seconds to completely dissolve the APTS.

Store at −20° C. for up to one year.

Derivatization Solution II (1 M acetic acid and 0.25 M NaBH3CN)

Dilute 20 4 acetic acid with 320 μL HPLC grade water (17 fold dilution)in a 0.4 mL centrifuge tube to make a 1 M acetic acid solution.

Weigh 2.0±0.5 mg of NaBH₃CN into a cryogenic vial.

Using the following formula, add an appropriate volume of the 1 M aceticacid solution to make 0.25 M NaBH₃CN. Volume (μL)=10₃×(weight of NaBH₃CNin mg)/(62.84 g/mol×0.25 mol/L)

• Sodium cyanoborohydride (NaBH₃CN) should be stored in dark in adesiccator. • Subdividing of the reagent into a series of 2.0 mLcryovials for storage is recommended to avoid repeated opening of theoriginal reagent bottle as follows: • Weigh 1.0 g±0.2 mg of SodiumCyanoborohydride into 2.0 mL cryovial. Aliquot out the entire contentsof Sodium Cyanoborohydride from the original bottle in this manner. •Cap tightly and label cryovials sequentially (1,2,3, etc.) along withreagent name, lot number, and a 6 month expiration date. • The vialsshould be sealed with parafilm to avoid moisture. • Weigh out SodiumCyanoborohydride for Derivatization Solution II no more than three timesfrom the same cryovial. Make note of this and the cryovial sequencenumber on the lab worksheet. • Either a reagent peak observed in the CEprofile or poor labeling may occur after repeated opening of thecryovial or with that particular lot of Sodium Cyanoborohydride. If thiseffects the results, discard the cryovial being used and either weighout reagent from a cryovial with the next sequence number or from a newlot of Sodium Cyanoborohydride.

Re-acetylation Buffer (25 mM sodium bicarbonate, pH 9.5)

Weigh 0.210±0.02 g of sodium bicarbonate into a clean 100 mL clean glassbeaker.

Add 90 mL of HPLC grade water, and mix on a stir plate until salts arecompletely dissolved.

Adjust the pH to 9.5±0.1 with 10 N NaOH.

Add HPLC grade water to make the final volume 100 mL. Filter (step 1.26)the solution and store at room temperature for up to 3 months.

Running Buffer (60±5 mM Sodium tetraborate, pH 9.25)

Weigh 1.21±0.02 g sodium tetraborate into a 100 mL clean glass beaker.

Add 90 mL of HPLC grade water, and mix on a stir plate until salts arecompletely dissolved.

Adjust the pH to 9.25±0.10 with 10 N NaOH.

Add HPLC grade water to make the final volume 100 mL for a finalconcentration of 60±5 mM.

For a 55 mM solution, weigh 1.11 g (±0.02) sodium tetraborate and followabove instructions for dissolving and titrating.

For a 65 mM solution, weigh 1.31 g (±0.02) sodium tetraborate and followabove instructions for dissolving and titrating.

Store at room temperature for up to 3 months. Prepare fresh buffer ifpeak resolution is effected (R value <1.0).

Optional: Dilute tetraborate buffer solution (MicroSolv) by adding 120mL of ultra pure water to 80 mL of 150 mM sodium tetraborate buffer fora final concentration of 60 mM (±5 mM). Titrate with 10N NaOH to bringthe solution pH to 9.25 (±0.1).

For a 55 mM tetraborate solution, dilute 66 mL of 150 mM sodiumtetraborate buffer into 114 mL of ultra pure water. Titrate as above.

For a 65 mM tetraborate solution, dilute 78 mL of 150 mM sodiumtetraborate buffer into 102 mL of ultra pure water. Titrate as above.

Store the solution at room temperature for a maximum of 3 months.Prepare fresh buffer if peak resolution is effected (R value <1.0).

Capillary Rinsing Solutions

N NaOH solution

Add 1 mL of 10 N NaOH solution to a 15 mL graduated plastic tubecontaining 9 mL of HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months.

N HCl solution:

Add 1 mL of 6 N HCl solution to a 15 mL graduated plastic tubecontaining 5 mL of HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months. 3.6.3 80%methanol solution:

Add 8 mL HPLC grade methanol to a 15 mL graduated plastic tubecontaining 2 mL HPLC grade water. Mix well by vortexing 5-10 sec.

Store the solution at room temperature for up to 6 months.

Monosaccharide Standard Stock Solutions

N-Acetyl Glucosamine (GalNAc)

Accurately weigh 5±1 mg of GalNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved.

Record the accurate concentration of the solution (mg/mL).

N-Acetyl Galactosamine (GlcNAc)

Accurately weigh 5±1 mg of GlcNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved.

Record the accurate concentration of the solution (mg/mL).

N-Acetyl Mannosamine (ManNAc)

Accurately weigh 5±1 mg of ManNAc into a 2.0 mL cryogenic vial.

Add 1 mL of HPLC grade water and mix well by vortexing until dissolved.

Record the accurate concentration of the solution (mg/mL).

Store Monosaccharide Standard Stock Solutions at −20° C. for up to 1year.

Monosaccharide Working Solution I: Internal Standard Working Solution

Dilute stock solution of ManNAc 100 fold with HPLC grade water by adding20 μL of ManNAc stock solution into a 2 mL cryogenic vial which alreadycontains 1980 μL of HPLC grade water. Vortex approximately 5 to 10seconds.

Store the internal standard working solution at 2-8° C. for up to 6months.

Monosaccharide Working Solution II: Amino Mix Standard Working Solution

In a 2.0 mL cryogenic vial containing 1960 μL of HPLC grade water, add20 μL of stock solutions of GalNAc and GlcNAc, respectively. Vortexapproximately 5 to 10 seconds.

Store the amino mix standard working solution at 2-8° C. for up to 6months.

Sample and reference material solutions.

Thaw frozen protein samples at 2-8° C., and gently mix by inversion.

Dilute both samples and reference material with HPLC grade water toabout 1.0 mg/mL. Make note of concentration out to three significantfigures.

CE Running Conditions

-   Running Buffer (step 2.5) 60 mM sodium tetraborate, pH 9.25-   Capillary Cartridge temperature 25° C.-   Voltage 25-30 kV, positive mode-   Detector condition LIF detector, Excitation: 488 nm, Emission: 520    nm.-   Sample injection Pressure injection mode, 20 s at 0.5 PSI-   Run Time 10 minutes-   Sample storage 10° C.

Procedure

Note: Use a 10 μL Pipettor and micro tips to transfer 10 μL samplevolumes and appropriately sized Pipettors to transfer other reagents(see ranges in steps 2.10 through 2.14).

Hydrolysis

In a 0.65 mL centrifuge tube, add 10 μL of ManNAc working solution and200 μL 4 N Hydrolysis Solution (step 3.1). This serves as a systemblank.

In a 0.65 mL centrifuge tube, add 10 μL of ManNAc working solution and10 μL of Amino Mix Standard Solution (step 3.9). Further add 200 μL of4N Hydrolysis Solution. This serves as monosaccharide standard forquantitation and System Suitability. Prepare in duplicate.

In a 0.65 mL centrifuge tube, add 10 μL of ManNAc working solution and10 μL of CTLA4-Ig reference material solution (approximately 1 mg/mL).

Further add 200 μL of 4N HCl solution. Prepare in duplicate.

In a 0.65 mL centrifuge tube, add 10 μL of ManNAc working solution and10 μL of sample solution (approximately 1 mg/mL). Further add 200 μL of4N HCl solution. Prepare in duplicate.

Vortex samples for approximately 10 seconds and centrifuge forapproximately 5-10 seconds. Place samples in a 96-position vial rack andincubate in an oven at 95° C. for 6 hr.

After hydrolysis, place hydrolyzed samples at −20° C. for 10 minutes tocool down.

Briefly centrifuge the hydrolyzed samples until any condensate is forcedto the bottom of the tube (5-10 seconds at high speed). Evaporatesamples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to “Low”.

Reconstitute each sample with 100 μL of HPLC grade water and vortex10-15 sec. Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to “Low”.

Re-acetylation

Reconstitute each sample with 10 μL of M6 re-acetylation buffer andvortex 5-10 sec. to mix well. Add 4 μL of M3 re-acetylation reagent intoeach tube. Vortex for approximately 5-10 seconds. Incubate on ice for 30minutes.

Note: The re-acetylation buffer (M6) and reagent (M3) can be replacedrespectively with 25 mM NaHCO₃ (add 20 μL) prepared in house and aceticanhydride (add 4 μL).

Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to “Low”.

Reconstitute each sample with 100 μL of HPLC grade water and vortex10-15 sec.

Evaporate samples to dryness in SpeedVac.

Note: Turn off SpeedVac heat, and set the evaporating rate to “Low”.

Derivatization

Place the micro centrifuge in the oven to equilibrate to the oventemperature of 55° C.

Reconstitute each sample with 10 μL of Derivitization Solution I (0.1 MAPTS solution, step 3.2). Vortex approximately 5-10 seconds.

Add 5 μL of the Derivatization Solution II (1M HAc and 0.25 M NaBH₃CN,step 3.3). Vortex approximately 5-10 seconds and centrifuge.

Quickly load the sample vials into the pre-warmed centrifuge, and placethe centrifuge back in the 55° C. oven. Incubate for 3 hr whilecentrifuging at 2000 rpm. This prevents the condensation of solvent onvial surface.

Instrumentation Preparation

Installing a new capillary, rinse in high pressure mode (80 PSI) usingthe following steps:

1 N NaOH for 20 minutes.

HPLC grade water for 10 minutes.

60 mM sodium tetraborate buffer for 10 minutes.

Operation

Before each operation, run the washing/rinse sequences to rinse thecapillary.

Then run the System Suitability Standard (monosaccharide standard) toensure the system is suitable.

Using 1N NaOH may etch the inside of capillaries from different vendorsand cause a shift in migration times throughout the run. If this causesthe migration time of the last peak (G1cNAc) to be more than 10.0minutes, it may be necessary to replace 1N NaOH with 0.1N NaOH or HPLCgrade water for the step 2 rinse.

When using an equivalent capillary and the above washing procedure isnot adequate using 80% methanol and/or 1N HCl may be necessary for thelast peak (G1cNAc) to be within the exemplary values of 10.0 minutes.

Preparation for injection

After derivatization, let samples cool down to room temperature.Centrifuge approximately 10 seconds at room temperature, untilcondensate is forced to the bottom of the tube.

Add 85 μL of HPLC grade water to each tube to bring the final volume ofeach sample to 100 μL. Vortex for 5-10 seconds.

Transfer 10 μL of sample from each tube to a CE micro vial and add 190μL of HPLC grade water to each tube. Vortex for 5-10 seconds.

Rinse steps and Injection sequence:

Note: For every four injections, change the CE running buffer with newlyprepared CE running buffer (due to ionic depletion effect). Performcapillary rinse at 40 psi.

System Suitability

Note: System suitability values are determined using the first injectionof system suitability standard unless otherwise specified.

The electropherogram of the first system suitability should be similarto that shown in FIG. 80, where peak 1 is GalNAc; peak 2 is ManNAc; peak3 is GlcNAc.

Note: When CE instruments other than Beckman PACE MDO are to be used,the length of the capillary might be different from that specified inthis method due to various configurations of cartridges holding theseparation capillary. This would cause variations in analyte migrationtime, as well as peak intensity.

Resolution between two neighbor peaks is calculated for the first SystemSuitability standard by the instrument according to the followingequation:

$(R) = \frac{2\left( {t_{2} - t_{1}} \right)}{W_{1} + W_{2}}$

-   -   Where:    -   t₁, t₂=migration times of the two neighbor peaks respectively    -   W₁, W₂=peak widths at baseline of the two neighbor peaks        respectively

The R value must be ≥1.0. If R <1.0, rinse the capillary with thewashing/rinse sequences; if the problem persists, replace old bufferwith freshly prepared Running Buffer or replace the capillary. For thelast System Suitability injection, the last peak (GlcNAc) must have atailing factor <1.4 using the following formula:

(T)=W_(0.05)/2f

-   -   Where:    -   T=tailing factor    -   W_(0.05)=width of peak at 5% of height    -   f=width of the peak front at peak maximum        If T ≥1.4, rinse the capillary with the washing/rinse sequences;        if the problem persists, replace old buffer with freshly        prepared run buffer or replace the capillary.

The replicate injections show the following exemplary values:

-   Peak Area Ratio of GlcNAc vs. MaNAc: RSD 10% (calculated in step    7.1)-   Migration time of GlcNAc should be 10.0 minutes-   Profile should be equivalent to FIG. 80 where the three peaks are    observed and the Internal Standard (ManNAc) is the number 2 peak.

If any of the above exemplary values are not reached prior to testingsamples, first increase the voltage if the migration time of GlcNAc isgreater than 10.0 minutes. Next, if the peak area ratio is >10%, preparefresh CE buffer making certain of its pH or replace the capillary. Afteradjustment to the instrument, repeat System Suitability injections. Whenanalyzing the peak profile, if a significant decrease in the peak heightof ManNac occurs, check to make certain the fiber optic cable into theLIF module is not misaligned.

Determine monosaccharide standard percent RSD by comparing peak arearatios of internal standard and monosaccharide standard components.Divide the peak area for each monosaccharide component by the peak areaof the internal standard for each monosaccharide standard injection.Calculate the percent RSD for GalNAc and GlcNAc for the two bracketedstandards. The RSD should be ≤10%. If this averaging exemplary value isnot met, then the capillary should be rinsed or replaced as above.

Calculations

Calculating Peak Area Ratio of GalNAc and GlcNAc relative to theInternal Standard (ManNAc). Used on replicate injections of first fourSystem Suitability Standards so as to meet above exemplary values andperforming same calculations on all of the bracketed, System SuitabilityStandards injected before and after sample(s).

Peak Area Ratio=Divide the peak area for each monosaccharide component(GlcNAc, GalNAc) by the peak area of the internal standard (ManNAc) foreach System Suitability Standard injection.

${{Peak}\mspace{14mu} {Area}\mspace{14mu} {Ratio}} = \frac{{monosaccharide}\mspace{14mu} {peak}\mspace{14mu} {area}}{{MaNAc}\mspace{14mu} {peak}\mspace{14mu} {area}}$

Calculate a mean of the Peak Area Ratios for GlcNAc and GalNAc in theSystem Suitability Standards. Also calculate a Standard Deviation (S.D.)and percent relative standard deviation (% RSD)

Exemplary Values: RSD for the Peak Area

Ratio of GlcNAc ≤10%.

Two, bracketed, System Suitability Standards injected before and aftersample(s): Percent RSD for the Peak Area Ratio of GlcNAc and GalNAc≤10%. If this averaging exemplary value is not met (RSD >10%), then thecapillary needs to be re-rinsed with the rinse procedures and thosesamples and bracketed monosaccharide standards need to be run again. Ifthe averaging exemplary value is still not met, replace the capillaryand rinse. Run the samples and bracketed monosaccharide standards again.

${{Standard}\mspace{14mu} {Deviation}} = \sqrt{\frac{{n{\sum x^{2}}} - \left( {\sum x} \right)^{2}}{n\left( {n - 1} \right)}}$

-   -   Where:    -   n=number of measurements in the sample    -   x=individual measurements

${\% \mspace{11mu} {RSD}} = {\frac{{Standard}\mspace{14mu} {Deviation}}{{Average}\mspace{14mu} {Measured}\mspace{14mu} {Peak}\mspace{14mu} {Area}} \times 100}$

Calculate the molar ratio of GalNAc/Protein:

$R_{GalNAc} = \frac{A_{GalNAc} \times A_{{ManNAc}\; 0} \times V_{{GalNAc}\; 0} \times C_{{GalNAc}\; 0} \times {MW}_{Abatacept}}{A_{{ManNAc}\;} \times A_{{GalNAc}\; 0} \times {Vp} \times {Cp} \times M\; W_{GlcNAc}}$

-   -   Where:    -   R_(GalNAc)=molar ratio of GalNAc vs. protein    -   A_(GalNAc)=peak area (μV·sec) of GalNAc in sample    -   A_(ManNAc)=peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0)=peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(GalNAc0)=peak area (μV·sec) average of GalNAC in        monosaccharide standard    -   V_(GalNAc0)=volume of GalNAc contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(GalNAc0)=conecntration of GalNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(Abatacept)=Molecular weight of Abatacept Reference Material        as per Certificat of Analysis (COA)    -   MW_(GlcNAc)=Molecular weight of GalNAc (221.2 daltons)

Standards Bracketing

When calculating molar ratios of CTLA4-Ig material and samples, use alleight of the bracketed System Suitability Standards. Average the peakareas for inclusion in this equation. This is to be used for the firstthree samples. For all other samples, always use the average peak areaof the next four bracketed monosaccharide standards and the previousfour bracketed monosaccharide standards for molar ratio calculations.

Calculate the molar ratio of GIcNAc/Protein

-   -   Where:    -   R_(GalNAc)=molar ratio of GIcNAc vs. protein    -   A_(GalNAc)=peak area (μV·sec) of GlcNAc in sample    -   A_(ManNAc)=peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0)=peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(GlcNAc0)=peak area (μV·sec) average of GlcNAc in        monosaccharide standard    -   V_(GlcNAc0)=volume of GlcNAc contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(GlcNAc0)=concentration of GlcNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(Abatacept)=Molecular weight of CGLA4-Ig Reference Material    -   MW_(GlcNAc)=Molecular weight of GlcNAc (221.2 daltons)

Exemplary values. The percent RSD for the two, bracketed, amino SystemSuitability Standard peak area ratios should not exceed 10%. The averagemolar ratios for amino monosaccharides in the reference material shouldbe within the ranges specified in the Table directly below. For eachcomponent, the % RSD for the four results (duplicate injection ofduplicate preparations) must be </=25%.

TABLE Molar Ratio range of CTLA4-Ig Reference Material MonosaccharideRange GAlNAc 2.0-3.2 GlcNAc 18-32

Example 17 Determination of Molar Ratio of Amino monosaccharides (GalNAcand GlcNAc) by Capillary Electrophoresis (CE)

In one embodiment, the CTLA4-Ig composition has the characteristic ofhaving from about 15-35 moles GlcNAc/mole of protein and from about1.7-3.6 moles GalNac/moles protein. The following example describes amethod of determining these molar ratios.

Reagents: Hydrolysis solution (4N HCl); Derivatization solution I (0.1M8-amino-1,3,6, trisulfonic acic, trisodium salt (APTS) aqueoussolution); Derivatization solution II (0.25M NaBH₃CN in 1M acetic acid);Re-acetylation buffer (25 mM sodium bicarbonate, pH9.5); Running buffer(60±5 mM sodium tetraborate, pH9.25); Capillary rinsing solutions (1NNaOH; 1N HCl; 80% methanol); Monosaccharide standard stock solutions ofGalNAc, GlcNAc, and ManNAc at concentration of 5 mg/ml; Monosaccharideworking solution I: Internal standard working solution is 100 folddilution of ManNAc stock solution; Monosaccharide working solution II:Amino mix standard working solutions, 100 fold dilution of GalNAc andGlcNAc stock solutions.

Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector:Beckman laser induced (LIF) detection system coupled with P/ACE MDQ);Uncoated capillary (i.d. 25 μm, o.d. 360 μm) 27-31 cm total length toaccommodate P/ACE MDQ. Capillary Electrophoresis running conditions:Running buffer (60 mM sodium tetraborate, pH 9.25); Capillary cartridgetemperature: 25° C.; Voltage: 25-30 kV, positive mode; Detectorcondition: LIF detector, excitation at 488 nm, emission at 520m; Sampleinjection: pressure injection mode, 20s at 0.5PSI; Run time: 10 min;Sample storage: 10° C.

Hydrolysis: 10 μL of ManNAc working solution and 200 μL of 4N HCl weremixed to make the system blank. 10 μL of ManNAc working solution and 10μof Amino mix standard solution were mixed with 200 μL of 4N HCl to makethe monosaccharide standard. 10 μL of ManNAc working solution and 10 μLof CTLA4-Ig dimer (approximately 1 mg/ml) were mixed with 200 μL of 4NHCl to make the test sample. All tubes were vortexed for 10 sec, andcentrifuge for 10 sec, followed by incubation at 95° C. for 6 hours.After the hydrolysis step the samples were places at −20° C. for 10 minto cool down. Samples were spun down for 10 sec and evaporated todryness in SpeedVac.

Re-acetylation: Hydrolyzed and dried samples were reconstituted with 100μL of HPLC grade water. Reconstituted samples were re-acetylated byaddition of 10 μL of M6 re-N-acetylation buffer (Glyko) and 4 L of M3re-acetylation reagent (Glyko), followed by mixing and with incubationon ice (30 min). Samples were spun down for 10 sec and evaporated todryness in SpeedVac.

Derivatization: Reconstituted samples (100 μL of HPLC grade water) wereequilibrated 55° C., followed by addition of 10 μL of Derivatizationsolution I, a brief mix, and addition of 5 μL of Derivatization solutionII. Samples were loaded in a pre-warmed centrifuge and incubated for 3hours at 55° C. while centrifuging at 2000 rpm.

CE injection: The final volume of the samples after derivatization wasbrought to 100 μL by addition of HPLC grade water, and 10 μL of sampleswere transferred to a CE micro vial with 190 μL HPLC grade water. Beforesample injections the CE cartridge was rinsed extensively with HPLCgrade water (1-3 min run time), followed by an equilibrating rinse withrunning buffer (5 min run time). Following the initial rinse,monosaccharide standards and samples for analysis were injected in theCE cartridge (10 min run time). Following the injection run of eachstandard or test sample, the CE cartridge was rinsed and equilibratedwith HPLC grade water and running buffer. The electopherograpm of thesystem suitability should be similar to FIG. 29.

Calculations: Calculating peak area ratio of GalNAc and GLCNAc relativeto internal standard ManNAc.

Peak area ratio=monosaccharide peak area (GalNAc or GlcNAc)/ManNAc peakarea,

-   wherein the relative standard deviation (RSD) for the peak area    ratio is equal or less that 10%.-   Calculating ratio of monosaccharide (for example GalNAc) to CTLA4-Ig    protein:

Ratio_(GalNAc)=(A_(GalNAc)×A_(ManNAcO)×V_(GalNAcO)×C_(GalNAcO)×MW_(CTLA4-IG dimer))/(A_(ManNAc)×A_(GalNAcO)×Vp×Cp×MW_(GalNAc))

-   Ratio_(GalNAc)=molar ratio of GalNAc versus protein-   A_(GalNAc)=peak area (μV·sec) in GalNAc sample-   A_(ManNAc)=peak area (μV·sec) in ManNAc sample-   A_(ManNAcO)=peak area (μV·sec) average of ManNA in monosaccharide    standard-   A_(GalNAcO)=peak area (μV·sec) average of GalNAc in monosaccharide    standard-   V_(GalNAcO)=volume of GalNAc contained in monosacchride working    solution used for hydrolysis (in μL)-   C_(GalNAcO)=concentration of GalNAc contained in monosacchride    working solution used for hydrolysis (in mg/ml)-   Vp=volume of protein sample used for hydrolysis (in μL)-   Cp=concentration of protein sample used for hydrolysis (in mg/ml)-   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig dimer-   MW_(GalNAc)=221.2 daltons.

TABLE 18 Average Molar Ratio of Monosaccharide to CTLA4-Ig molecules ordimer MONOSACCHARIDE RANGE GalNAc 2.0-3.2 GlcNAc 18-32

Example 18 Determination of Molar Ratio of Neutral Monosaccharides(Mannose, Fucose and Galactose) by Capillary Electrophoresis (CE)

Reagents: Hydrolysis solution (2M trifluoroacetic acid (TFA));Derivatization solution I (0.1M 8-amino-1,3,6, trisulfonic acic,trisodium salt (APTS) aqueous solution); Derivatization solution II(0.25M NaBH₃CN in 1M acetic acid); Running buffer (60±5 mM sodiumtetraborate, pH9.25); Capillary rinsing solutions (1N NaOH; IN HCl; 80%methanol); Monosaccharide standard stock solutions of mannose (Man),fucose (Fuc), galactose (Gal), and xylose (Xyl) at concentration of 5mg/ml; Monosaccharide working solution I: Internal standard workingsolution is 100 fold dilution of Xyl stock solution; Monosaccharideworking solution II: Neutral mix standard working solutions, 100 folddilution of Man, Fuc and Gal stock solutions.

Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector:Beckman laser induced (LIF) detection system coupled with P/ACE MDQ);Uncoated capillary (i.d. 25 μm, o.d. 360 μm) 27-31 cm total length toaccommodate P/ACE MDQ.

Capillary Electrophoresis running conditions: Running buffer (60 mMsodium tetraborate, pH 9.25); Capillary cartridge temperature: 25° C.;Voltage: 25-30 kV, positive mode; Detector condition: LIF detector,excitation at 488 nm, emission at 520m; Sample injection: pressureinjection mode, 20s at 0.5 PSI; Run time: 10 min; Sample storage: 10° C.

Hydrolysis: 10 μL of Xylose working solution and 200 μL of 2M TFA weremixed to make the system blank. 10 μL of Xylose working solution and 10μL of Neutral mix standard solution were mixed with 200 μL of 2M TFA tomake the monosaccharide standard. 10 μL of Xylose working solution and10 μL of CTLA4-Ig dimer (approximately 1 mg/ml) were mixed with 200 μLof 2M TFA to make the test sample. All tubes were vortexed for 10 sec,and centrifuge for 10 sec, followed by incubation at 95° C. for 6 hours.After the hydrolysis step the samples were places at −20° C. for 10 minto cool down. Samples were spun down for 10 sec and evaporated todryness in SpeedVac.

Derivatization: Samples were reconstituted with 100 μL of HPLC gradewater and were equilibrated 55° C., followed by addition of 10 μL ofDerivatization solution I, a brief mix, and addition of 54 ofDerivatization solution II. Samples were loaded in a pre-warmedcentrifuge and incubated for 3 hours at 55° C. while centrifuging at2000 rpm.

CE injection: The final volume of the samples after derivatization wasbrought to 100 μL by addition of HPLC grade water, and 10 μL of sampleswere transferred to a CE micro vial with 190 μL HPLC grade water. Beforesample injections the CE cartridge was rinsed extensively with HPLCgrade water (1-3 min run time), followed by an equilibrating rinse withrunning buffer (5 min run time). Following the initial rinse,monosaccharide standards and samples for analysis were injected in theCE cartridge (15 min run time). Following the injection run of eachstandard or test sample, the CE cartridge was rinsed and equilibratedwith HPLC grade water and running buffer. The electopherograpm of thesystem suitability should be similar to FIG. 30.

Calculations: Calculating peak area ratio of Man, Gal and Fuc relativeto internal standard Xylose.

Peak area ratio=monosaccharide peak area (Gal, Fuc or Man)/Xylose peakarea, wherein the relative standard deviation (RSD) for the peak arearatio is equal or less that 10%.

-   Calculating ratio of monosaccharide (for example Man) to CTLA4-Ig    protein:

Ratio_(Man)=(A_(Man)×A_(XylO)×V_(ManO)×C_(ManO)×MW_(CTLA4-Ig dimer))/(A_(Xyl)×A_(ManO)×Vp×Cp×MW_(Man))

-   Ratio_(Man)=molar ratio of Man versus protein-   A_(Man)=peak area (μV·sec) in Man in sample-   A_(Xyl)=peak area (μV·sec) in Xy1 in sample-   A_(XylO)=peak area (μV·sec) average of Xy1 in monosaccharide    standard-   A_(ManO)=peak area (μV·sec) average of Man in monosacchardie    standard-   V_(ManO)=volume of Mannose contained in monosacchride working    solution used for hydrolysis (in μL)-   C_(ManO)=conecntration of Mannose contained in monosacchride working    solution used for hydrolysis (in mg/ml)-   Vp=volume of protein sample used for hydrolysis (in μL)-   Cp=concentration of protein sample used for hydrolysis (in mg/ml)-   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig dimer-   MW_(MAN)=180.2 daltons.

TABLE 19 Average Molar Ratio of Monosaccharide to CTLA4-Ig molecules ordimer MONOSACCHARIDE RANGE Mannose 10-20 Fucose 4.2-7.0 Galactose9.2-17 

Example 19 Production of CTLA4^(A29YL104E)-Ig

CTLA4^(A29YL104E)-Ig is a genetically engineered fusion protein, whichconsists of the functional binding domain of modified human CTLA-4 andthe Fc domain of human immunoglobulin of the IgGl class. Two amino acidsubstitutions were made in the B7 binding region of the CTLA-4 domain(L104E and A29Y) to generate this molecule. It is comprised of twoglycosylated polypeptide chains of 357 amino acids each. It exists ascovalent dimer linked through an inter-chain disulfide bond.CTLA4^(A29YL104E)-Ig has an average mass of approximately 91,800 Da asdetermined by matrix-assisted laser desorption-ionization time-of-flight(MALDI-TOF) mass spectrometry.

CTLA4^(A29YL104E)-Ig is a modified form of CTLA4-Ig. The modificationconsists of point mutations that result in two amino acid substitutions(L104E and A29Y). Relative to CTLA4-Ig, CTLA4^(A29YL104E)-Ig binds CD80(B7-1) with ˜2-fold increased avidity, and binds CD86 (B7-2) with˜4-fold increased avidity. CTLA4^(A29YL104E)-Ig is approximately 10-foldmore effective than abatacept at inhibiting T cell proliferation,cytokine production, and CD28-dependent killing of target cells bynatural killer cells. CTLA4^(A29YL104E)-Ig causes modest inhibition ofB7-1 mediated T cell proliferation but is markedly more potent atblocking B7-2 mediated T cell proliferation. This Example describes theproduction of CTLA4^(A29YL104E)-Ig molecules comprising SEQ ID NO:4. Themethods described in this Example can be adapted and extended for theproduction of other recombinant proteins, including but not limited to,secreted proteins such as cytokines and other hormones, secretedproteins that are members of the Ig superfamily or comprise a portion ofan Ig superfamily protein, and generally any protein expressed in CHOcells.

A process flow diagram for the CTLA4^(A29YL104E)-Ig culturing steps isshown in FIG. 23. CTLA4^(A29YL104E)-Ig is produced in 5000-L productionbioreactors with an approximate working volume of 4000 L. One batch ofdrug substance is produced from a single production bioreactor derivedfrom a single vial from a cell bank. The production process involvesthree-stages consisting of inoculum expansion, production cell cultureand downstream purification. The inoculum expansion stage is conductedusing animal component-free medium. The production cell culture stage isalso performed in animal component-free medium with the exception of theuse of D-galactose.

Cell Culture Media. All media are prepared in clean medium vessels ofthe appropriate size and sterilized by filtration. The composition ofthe medium utilized for inoculum expansion is presented in the Tablebelow.

Inoculum Cell Growth Basal Medium

Component Concentration CD-CHO, 25x Concentrate Acid Solubles I 40 mL/LCD-CHO, 25x Concentrate Acid Solubles II 40 mL/L CD-CHO, 25x ConcentrateSalts I 40 mL/L CD-CHO, 25x Concentrate Salts II 40 mL/L L-glutamine0.88 g/L Sodium Bicarbonate 2.22 g/L Recombinant Human Insulin (10mg/mL) 0.1 mL/L Methotrexate (20 mM) 0.05 mL/L

Seed and Production Bioreactor Cell Growth Basal Medium

Component Concentration CD-CHO, 25x Concentrate Acid Solubles I 40 mL/LCD-CHO, 25x Concentrate Acid Solubles II 40 mL/L CD-CHO, 25x ConcentrateSalts I 40 mL/L CD-CHO, 25x Concentrate Salts II 40 mL/L L-glutamine1.32 g/L Sodium Bicarbonate 2.22 g/L Recombinant Human Insulin (10mg/mL) 0.1 mL/L

Production Bioreactor Feed Medium

Component Concentration eRDF powder^(a) 25.2 g/L Dextrose 30.9 g/LD-galactose 12.5 g/L L-glutamine 4.1 g/L Recombinant Human Insulin (10mg/mL) 1.0 mL/L Dextran Sulfate (added as bolus feed) 50 mg/L

Inoculum Expansion

A frozen vial from the cell bank is thawed at a controlled temperatureand centrifuged to remove the cryoprotectant media. The cells areresuspended in inoculum medium and recovered in a T-flask. A minimumcell viability after thaw of 80% is an exemplary value. Temperature andcarbon dioxide are controlled during the T-flask incubation step. TheT-flask is incubated until a viable cell number of 1.0×10⁷ cells isobtained, and the contents are transferred into a shake flask. Theculture is expanded through a series of shake flasks to achieve therequired inoculum volume. The seeding density range for the shake flaskpassages is 1.0 to 3.0×10⁵ viable cells/mL. Temperature, carbon dioxide,and shaker speed are controlled during the shake flask incubation steps.The shake flask cultures are pooled into a sterile inoculum transfervessel upon reaching a viable cell density range of 1.5 to 3.0×10⁶cells/mL. Approximately 20 liters from the final shake flask inoculumexpansion step is transferred to the 140-L seed bioreactor to achieve aninitial cell density range of 0.2 to 1.0×10⁶ viable cells/mL.

Seed Bioreactor Operation

A 140-L seed bioreactor with a working volume of approximately 90 litersis operated in batch mode. The temperature, pH, pressure, and dissolvedoxygen concentration in the 140-L seed bioreactor are monitored andcontrolled using a distributed control system (DCS). Samples areobtained daily from the 140-L seed bioreactor to monitor cell growth.The seeding density range of the 140-L seed bioreactor is 0.2 to 1.0×10⁶viable cells/mL. The 140-L seed bioreactor culture is used to inoculatea 1100-L seed bioreactor when a viable cell density of 1.5×10⁶ cells/mLis achieved. The duration of the 140-L seed bioreactor step isapproximately 3 days. The initial target viable cell density in the1100-L seed bioreactor is 0.4 to 1.5×10⁶ viable cells/mL.

The 1100-L seed bioreactor contains an initial culture volume of 260liters. The 1100-L seed bioreactor is operated in batch mode. Thetemperature, pH, pressure, and dissolved oxygen concentration in the1100-L seed bioreactor are monitored and controlled using a DCS. Thevolume of the culture is increased to 900 liters with basal medium whenthe viable cell density has reached ≥1.5×10⁶ cells/mL. Samples areobtained daily from the 1100-L seed bioreactor to monitor cell growth.The 1100-L seed bioreactor culture is used to inoculate a 5000-Lproduction bioreactor when a viable cell density of ≥2.0×10⁶ cells/mL isachieved. The duration of the 1100-L seed bioreactor step isapproximately 4 days. The initial target viable cell density in the5000-L production bioreactor is 0.4 to 1.5×10⁶ viable cells/mL.

Production Bioreactor Operation

The 5000-L production bioreactor contains an initial culture volume of3000 liters. The 5000-L production bioreactor is operated in fed-batchmode with temperature, pH, pressure, and dissolved oxygen concentrationmonitored and controlled using a DCS. A bolus of dextran sulfate isadded to the culture at approximately 72 hours. During the operation ofthe production bioreactor, the culture temperature setpoint is shiftedfrom 37° to 34° C. at 144±8 hours. The temperature shift and the dextransulfate addition are performed to prolong the duration of high cellviability in the 5000-L production bioreactor step. Samples are obtainedfrom the bioreactor to monitor cell growth and viability, glucose,lactate and ammonia concentration. The samples are also tested forCTLA4^(A29YL104E)-Ig concentration and sialic acid toCTLA4^(A29YL104E)-Ig protein molar ratio. The feed medium is added tothe bioreactor to maintain a desired glucose concentration. The primaryharvest criterion for the production bioreactor is the sialic acid toCTLA4^(A29YL104E)-Ig protein molar ratio. The production bioreactor isharvested at a target sialic acid to CTLA4^(A29YL104E)-Ig protein molarratio of ≥6. The duration of the 5000-L production bioreactor step isapproximately 14 days. The harvest volume of the 5000-L productionbioreactor is approximately 4000 liters.

Cell Removal and Product Concentration

Cells are removed from the culture broth by tangential flowmicrofiltration using 0.65 μm membranes. The microfiltration permeate isconcentrated by tangential flow ultrafiltration using 30 kDa nominalmolecular weight cutoff (NMWCO) membranes. Transmembrane pressure andflow rates are controlled during the microfiltration and ultrafiltrationsteps. The concentrate is then passed through a series of membranefilters, with a final filtration through a 0.2 μm single-use filter. ThepH of the concentrate is adjusted to 8.0 by the addition of a 0.5 M Trissolution. The microfiltration and ultrafiltration filters are multi-use.The microfiltration filters are cleaned with sodium hypochlorite andTriton X-100 and stored in phosphoric acid. The ultrafiltration filtersare cleaned with sodium hypochlorite and sodium hydroxide and thenstored in sodium hydroxide.

Example 20 Purification of Recombinant CTLA4^(A29YL104E)-Ig Example 20-A

An example of a purification process of CTLA4^(A29YL104E)-Ig is shown inthe flow diagram in FIG. 89. A description of a purification process isprovided by this example.

Viral Inactivation

The pH of the clarified concentrated harvest material is adjusted to 8.0by the addition of a 0.5 M Tris solution. Potential adventitious viralagents are inactivated by the addition of 20% Triton X-100 to a finalconcentration of 0.5% (v/v). The Triton X-100-treated protein solutionis mixed for ≥2 hours.

Affinity Chromatography

Affinity chromatography using a column of MabSelect Protein A resin (GEHealthcare, formerly known as Amersham Biosciences) is used to capturethe CTLA4^(A29YL104E)-Ig protein from the in-process material from theviral inactivation step and to separate the belatacept protein from themajority of impurities.

The MabSelect Protein A column is equilibrated with a 25 mM NaH₂PO₄, 150mM NaCl, pH 7.5 buffer. The dynamic binding capacity of the affinityresin is 25 g of CTLA4^(A29YL104E)-Ig protein per liter of resin at alinear velocity of 350 cm/hour. The 157-L column bed is capable ofbinding approximately 3.9 kg of CTLA4^(A29YL104E)-Ig protein.

The Triton X-100-treated in-process material is applied to the MabSelectProtein A column, and the column is washed with a minimum of 3 columnvolumes (CV) of equilibration buffer to remove weakly retainedimpurities. These impurities include the cytokine monocyte chemotacticprotein-1 (MCP-1) and Triton X-100. The CTLA4^(A29YL104E)-Ig protein isthen eluted from the column with a 250 mM glycine, pH 3.0 buffer. TheCTLA4^(A29YL104E)-Ig protein elutes as a narrow peak in approximately 2to 3 CV of elution buffer and is collected into a tank containing 2 MHEPES, pH 7.5 buffer in order to increase the pH rapidly and therebyminimize the formation of belatacept high molecular weight (HMW)species.

Anion Exchange Chromatography

Anion exchange chromatography using Q-Sepharose Fast Flow (QFF) resin(GE Healthcare) is used primarily to enrich the amount of more highlysialylated species of the CTLA4^(A29YL104E)-Ig protein. The pH-adjustedbelatacept product pool from the MabSelect Protein A column is dilutedapproximately two-fold with water for injection (WFI) prior toapplication to the QFF column.

The QFF column is equilibrated with a 50 mM HEPES, 50 mM NaCl, pH 7.0buffer. The pH-and conductivity-adjusted MabSelect Protein A stepproduct pool is applied to the QFF column, and the column is washed witha minimum of 3 CV of equilibration buffer to remove weakly boundimpurities. The column is then washed with 50 mM HEPES, 140 mM NaCl, pH7.0 buffer, to remove CTLA4^(A29YL104E)-Ig protein species with lowsialic acid content. The more highly sialylated species of theCTLA4^(A29YL104E)-Ig protein are subsequently eluted from the columnusing 5 CV of 50 mM HEPES, 200 mM NaCl, pH 7.0 buffer.

Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) using Toyopearl Phenyl 650Mresin (Tosoh Biosciences) is used primarily to reduce the amount ofCTLA4^(A29YL104E)-Ig HMW species in the product pool from the QFFchromatography step. Prior to application to the HIC column, the QFFchromatography step product pool is diluted using 50 mM HEPES, pH 7.0buffer and 50 mM HEPES, 3.6 M ammonium sulfate, pH 7.0 buffer to achievea conductivity of approximately 135 mS/cm and a CTLA4^(A29YL104E)-Igconcentration of 1 g/L in the QFF product pool.

The HIC column is equilibrated with a 50 mM HEPES, 1.2 M ammoniumsulfate, pH 7.0 buffer. The concentration-and conductivity-adjustedCTLA4^(A29YL104E)-Ig QFF chromatography step product pool is applied tothe column. The column is then washed with a 50 mM HEPES, 1.2 M ammoniumsulfate, pH 7.0 buffer to remove weakly bound impurities. TheCTLA4^(A29YL104E)-Ig protein is eluted from the HIC column using a 50 mMHEPES, 0.55 M ammonium sulfate, pH 7.0 buffer.

Viral Filtration

Concentration and diafiltration of the CTLA4^(A29YL104E)-Ig product poolfrom the HIC step is achieved by ultrafiltration (UF). The UF steputilizes a 30-kDa NMWCO membrane and a 25 mM NaH₂PO₄, 10 mM NaCl, pH 7.5buffer. The UF step is followed by a viral filtration step using a 15-nmPlanova membrane (Asahi Kasei). The CTLA4^(A29YL104E)-Ig protein productpool is then adjusted to a protein concentration of 25 g/L by UF using a30-kDa NMWCO membrane.

Column Sanitization and Storage

The MabSelect Protein A chromatography column is sanitized using 0.1 NNaOH solution, washed with 25 mM NaH₂PO₄, 150 mM NaCl, pH 7.5 buffer tolower the pH, and then stored in 20% ethanol at 2° to 8° C. The QFFchromatography column is sanitized with 1 N NaOH solution and stored in0.1 N NaOH solution at room temperature. The HIC column is sanitizedwith 0.1 N NaOH solution, washed with 20% ethanol, and stored in 20%ethanol at room temperature.

Example 20-B A Further Example of such Purification Method Follows

Viral Inactivation

The pH of the clarified concentrated harvest material is adjusted to 8.0by the addition of a 0.5 M Tris solution. Potential adventitious viralagents are inactivated by the addition of 20% Triton X-100 to a finalconcentration of 0.5% (v/v). The Triton X-100-treated protein solutionis mixed for 2 hours.

Protein A Affinity Chromatography for CTLA4^(A29YL104E)-Ig Purification:Affinity chromatography using a column of MabSelect Protein A resin (GEHealthcare, formerly known as Amersham Biosciences) is used to captureCTLA4^(A29YL104E)-Ig from the in-process material from the viralinactivation step and to separate CTLA4^(A29YL104E)-Ig from the majorityof impurities.

A 140 cm inner diameter column is packed with MabSelect PrA resin to aheight of 18 to 25 cm, representing a volume of about 339 to 372 L. Thecolumn is qualified for use by determining HETP and A_(s) of the packedcolumn. A HETP of 0.02 to 0.08 cm and an A_(s) of 0.8 to 1.2 areemployed for qualification of the column.

The MabSelect PrA column operation is carried out at ambienttemperature. The viral inactivation product pool is loaded onto theequilibrated MabSelect PrA column. The MabSelect PrA step is operated ata maximum flow rate of 26.7 L/min and an operating pressure of ≤13 psig.The maximum CTLA4^(A29YL104E)-Ig protein load applied to the MabSelectPrA column is 25 g of CTLA4^(A29YL104E)-Ig protein per liter of resin ata linear velocity of 350 cm/hour. The column bed is capable of bindingapproximately 3.9 kg of CTLA4^(A29YL104E)-Ig protein.

The MabSelect PrA column is equilibrated with a 25 mM NaH₂PO₄, 150 mMNaCl, pH 7.5 buffer. Equilibration is complete when a minimum of 3 CV ofequilibration buffer have been passed through the column and the pH andconductivity values of the effluent are between 7.3 to 7.7 and 14.5 to17.5 mS/cm, respectively.

The Triton X-100-treated in-process material is applied to theequilibrated MabSelect PrA column. The column is washed with a minimumof 3 CV of a 25 mM NaH₂PO₄, 150 mM NaCl, 0.5% Triton X-100, pH 7.5buffer to remove weakly retained impurities from the MabSelect PrAcolumn. These impurities include the cytokine monocyte chemotacticprotein-1 (MCP-1) and Triton X-100. Subsequent wash steps are performedusing a 25 mM NaH₂PO₄, 150 mM NaCl, pH 7.5 buffer to remove the residualTriton X-100 from the MabSelect PrA column.

The CTLA4^(A29YL104E)-Ig is eluted from the MabSelect PrA chromatographycolumn with a 250 mM glycine, pH 3.0 buffer. The eluate is diverted intoa collection vessel when the A₂₈₀ increases to ≥0.2 AU above thebaseline. The column effluent is filtered through a 0.2 μm celluloseacetate filter into a collection vessel equipped with an agitator. Theeluate is collected until the A₂₈₀ of the trailing edge of the elutionpeak decreases to a value of ≤0.2 AU. The CTLA4^(A29YL104E)-Ig elutes asa narrow peak in approximately 2 to 3 CV of elution buffer. The pH ofthe eluate pool is adjusted to pH 7.5±0.2 with a 2 M HEPES, pH 7.5buffer in order to increase the pH rapidly and thereby minimize theformation of CTLA4^(A29YL104E)-Ig high molecular weight (HMW) species.The MabSelect PrA chromatography step product pool is held at ambienttemperature for a maximum of 5 days. The product pool may be cooled forstorage; the stability profile of the CTLA4^(A29YL104E)-Ig was the sameat 5° C. and 22° C. The product may be stored for up to 5 days.

A CTLA4^(A29YL104E)-Ig dimer product with a molar ratio of moles sialicacid to moles CTLA4^(A29YL104E)-Ig protein that is about 6, or fromabout 5.2 to about 7.6, is collected.

QFF Anion Exchange Chromatography for CTLA4^(A29YL104E)-Ig Purification:Anion exchange chromatography using Q-Sepharose Fast Flow (QFF) resin(GE Healthcare) is used primarily to enrich the amount of more highlysialylated species of the CTLA4^(A29YL104E)-Ig as well as reduce theresidual Protein A levels. The pH-adjusted CTLA4^(A29YL104E)-Ig productpool from the MabSelect Protein A column is diluted approximatelytwo-fold with water for injection (WFI) prior to application to the QFFcolumn.

A 80 cm inner diameter column is packed with QFF resin to a height of 27to 35 cm, representing a volume of about 136 to 176 L. The column isqualified for use by determining the HETP and A_(s) of the packedcolumn. A HETP of 0.02 to 0.08 cm and an asymmetry (A_(s)) of 0.8 to 1.2are employed for qualification of the column.

The QFF column operation is carried out at ambient temperature. The QFFcolumn is equilibrated with a 50 mM HEPES, 50 mM NaCl, pH 7.0 buffer.The pH-and conductivity-adjusted MabSelect Protein A step product poolis applied to the QFF column. The QFF step is operated at a maximum flowrate of 16.4 L/min (196 cm/h) and a maximum operating pressure of 35psi.

The column is sanitized both prior to and following use with a 1 N NaOHsolution. A minimum of 2 column volumes of the sodium hydroxide solutionis passed over the column. The column is then held static for 60 to 120minutes. The acceptable conductivity range for the solution and thecolumn effluent is 136 to 202 mS/cm.

The column is equilibrated with a minimum of 5 column volumes of a 50 mMHEPES, 50 mM sodium chloride, pH 7.0 buffer. The pH and conductivityranges for this buffer are 6.8 to 7.2 and 5.0 to 7.0 mS/cm,respectively. These ranges are also used to determine whether the columnis equilibrated.

The pH-and conductivity-adjusted MabSelect Protein A step product poolis applied to the QFF column, and the column is washed with a minimum of3 CV of equilibration buffer to remove weakly bound impurities. Thecolumn is then washed with 50 mM HEPES, 135 mM NaCl, pH 7.0 buffer, toremove CTLA4^(A29YL104E)-Ig species with low sialic acid content.

The more highly sialylated species of the CTLA4^(A29YL104E)-Ig areeluted from the QFF chromatography column using a 50 mM HEPES, 200 mMNaCl, pH 7.0 buffer. The eluate collection is initiated when the elutionbuffer is first applied to the column. During elution, the columneffluent is filtered through a 0.2 μm filter into the collection vessel.The eluate is collected until the absorbance of the trailing edge of theelution peak decreases to ≤0.2 AU above the baseline. TheCTLA4^(A29YL104E)-Ig elutes from the column using ≤5 CV of 50 mM HEPES,200 mM NaCl, pH 7.0 buffer. The collection vessel is then cooled to 2°to 8° C. The maximum hold time for the QFF chromatography step productpool at 2° to 8° C. is 3 days.

A CTLA4^(A29YL104E)-Ig dimer product with a molar ratio of moles sialicacid to moles CTLA4^(A29YL104E)-Ig protein that is about 6, or fromabout 5.2 to about 7.6 is collected.

Phenyl Sepharose FF HIC for CTLA4^(A29YL104E)-Ig Purification:Hydrophobic interaction chromatography (HIC) using Toyopearl Phenyl 650Mresin (Tosoh Biosciences) is used primarily to reduce the amount ofCTLA4^(A29YL104E)-Ig HMW species in the product pool from the QFFchromatography step.

A 100 cm inner diameter column is packed with Phenyl Sepharose Phenyl650M resin to a height of 18 to 22 cm, representing a volume of about141 to 173 L. The column is qualified for use by determining the HETPand A_(s) of the packed column. A HETP of 0.02 to 0.08 cm and an A_(s)of 0.8 to 1.2 are employed for qualification of the HIC column.

The HIC column operation is carried out at ambient temperature. Prior toapplication to the HIC column, the QFF chromatography step product poolis diluted using 50 mM HEPES, pH 7.0 buffer and 50 mM HEPES, 3.6 Mammonium sulfate, pH 7.0 buffer to achieve a conductivity ofapproximately 135 mS/cm and a CTLA4^(A29YL104E)-Ig concentration of 1g/L in the QFF product pool. The HIC step is operated at a maximum flowrate of 22.7 L/min (173 cm/h) and at a maximum operating pressure of 45psi. Multiple cycles of the HIC step can be employed based on the amountof CTLA4^(A29YL104E)-Ig present in the QXL eluate pool.

The HIC column is first sanitized with a 1 N sodium hydroxide solution.The sanitization is complete when 2 to 4 CV of the 1 N sodium hydroxidesolution have been passed through the column. The column is then heldfor 60 to 120 minutes to ensure sanitization.

After the sanitization step, the HIC column is equilibrated with a 50 mMHEPES, 1.2 M ammonium sulfate, pH 7.0 buffer. The equilibration iscomplete when a minimum of 3 CV of equilibration buffer have been passedthrough the column and the pH of the effluent is 7.0±0.3 and theconductivity of approximately 135 mS/cm.

The concentration-and conductivity-adjusted CTLA4^(A29YL104E)-Ig QFFchromatography step product pool is applied to the column. The column isthen washed with a 50 mM HEPES, 1.2 M ammonium sulfate, pH 7.0 buffer toremove weakly bound impurities. The CTLA4^(A29YL104E)-Ig is eluted fromthe HIC column using a 50 mM HEPES, 0.55 M ammonium sulfate, pH 7.0buffer. This HIC product pool is held in the collection vessel at 2° to8° C. The maximum hold time in the collection vessel is 3 days.

A CTLA4^(A29YL104E)-Ig dimer product with a molar ratio of moles sialicacid to moles CTLA4^(A29YL104E)-Ig protein that is about 6, or fromabout 5.2 to about 7.6; a pool of CTLA4^(A29YL104E)-Ig high molecularweight material is present at ≤2.5%; a pool of CTLA4^(A29YL104E)-Ig lowmolecular weight material (for example CTLA4^(A29YL104E)-Ig monomer) ispresent at <0.5%; and a pool of MCP-1 <9.5 ng/mL is present.

Viral Filtration. Concentration and diafiltration of theCTLA4^(A29YL104E)-Ig product pool from the HIC step is achieved byultrafiltration (UF). The UF step utilizes a 30-kDa NMWCO membrane and a25 mM NaH₂PO₄, 10 mM NaCl, pH 7.5 buffer. The UF step is followed by aviral filtration step using a 15-nm Planova membrane (Asahi Kasei). TheCTLA4^(A29YL104E)-Ig product pool is then adjusted to a proteinconcentration of 25 g/L by UF using a 30-kDa NMWCO membrane.

The Pall Filtron TFF system is used in the concentration anddiafiltration step of the downstream CTLA4^(A29YL104E)-Ig productionprocess. The objective of this step is to concentrate the HICchromatography step product pool to 45 to 55 g/L and to exchange theelution buffer used in the HIC chromatography step with the final bufferused for CTLA4^(A29YL104E)-Ig compositions. The concentratedCTLA4^(A29YL104E)-Ig product pool is transferred through a 0.2 μmpolyvinylidene fluoride filter and into a 50-L bioprocess bag.

Example 21 Biological Activity—Determination of Bio-Specific Binding ofCTLA4A29YL104E-IG to the B-7IG Co-Receptor by Surface Plasmon Resonance

Surface Plasmon Resonance (B7 Binding)

This method measures the binding of CTLA4^(A29YL104E)-Ig to arepresentative B7 co-receptor by surface plasmon resonance. B7Ig isimmobilized at high density via primary amino groups to the surface ofan activated CM5 sensorchip. CTLA4^(A29YL104E)-Ig material, QualityControls, and samples are diluted to concentrations between 0.125 and 8ng/mL and injected over the B7Ig surface to generate bindingsensorgrams. The initial rate (slope) of CTLA4^(A29YL104E)-Ig binding toimmobilized B7Ig is measured under mass transfer (diffusion) limitedconditions on this B7Ig surface. The initial binding rate in resonanceunits per second (RU/s) correlates directly with the activeconcentration. The binding rates of samples are converted into an activeconcentration using the reference standard curve where the binding rateof a CTLA4^(A29YL104E)-Ig material is plotted against concentration. Thefinal results are either expressed as percent binding of sample relativeto CTLA4^(A29YL104E)-Ig material.

The presence of the human IgGl Fc region in CTLA4^(A29YL104E)-Ig wasdetected using surface plasmon resonance (SPR). SPR enables measurementof biospecific interactions in real time. An antibody fragment specificfor the Fc region of human IgG (goat F_((ab′)2) anti-human IgG Fc) wascovalently immobilized on the surface of a sensorchip. Binding ofCTLA4^(A29YL104E)-Ig samples was detected by measuring the responseobtained on this surface, compared to an unmodified sensor chip surface.The results in resonance units bound for the Process B, Process C andthe Co-mixture are comparable as shown in FIG. 6 and Table 23.

TABLE 23 Detection of Human IgG Fc in CTLA4^(A29YL104E)-Ig DrugSubstance Lots Using SPR RU bound to anti-Fc RU bound to unmodified LotNo Antibody surface Lot A 1295 1 Lot B 1309 1 Co-mixture 1268 1

Human Cell IL 2 Inhibition Assay

The method is based on the inhibition of IL-2 production from T cells byCTLA4^(A29YL104E)-Ig when stimulated with anti-CD3 and B cells. Jurkat Tcells, transfected with the luciferase gene under the control of theIL-2 promoter, are co-stimulated with Daudi B cells and anti-CD3 in thepresence of various concentrations of CTLA4^(A29YL104E)-Ig. Theco-stimulation activates the IL-2 promoter, which in turn producesluciferase protein. The resulting luminescent signal is measured using aluciferase assay system. In this system, CTLA4^(A29YL104E)-Ig produces adose-dependent decrease in luciferase activity.

The results for the Process B Lot 000929-278, Process C Lot224818-2004-007 and the co-mixture Lot 55128-162 are comparable as shownin Table 24. The EC₅₀ values, slope factors, and upper and lowerasymptotes are similar for all three samples, within one standarddeviation. This indicates that CTLA4^(A29YL104E)-Ig from the Process Cand from the Process B behave comparably in the in vitro Potency assay.

TABLE 24 Comparison of Human IL-2 Promoter Mediated Luciferase Activityin the In Vitro Potency Bioassay. Dose Response Curve Parameters forProcesses of the Invention Upper Lower EC₅₀ Asymptote Asymptote Lot No.(ng/mL) Slope Factor (CPS) (CPS) Process A 19.1 ± 1.9 −0.91 ± 0.0685,000 ± 15,000 30,000 ± 6,000 Co- 21.5 ± 2.7 −0.93 ± 0.08 88,000 ±16,000 29,000 ± 5,000 mixture Process B 21.8 ± 1.4 −0.91 ± 0.09 81,000 ±17,000 27,000 ± 7,000

Materials:

-   Sensor Chip CMS, certified grade Biacore (Catalog No. BR-1000-13)-   HBS-EP Buffer BIA Certified 10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM    EDTA, 0.005% v/v-   Surfactant P20 Biacore (Catalog No. BR-1001-88)-   Amine Coupling Kit BIA Certified115 mg N-hydroxysuccinimide (NHS),    750 mg 1-ethyl-3-(3 dimethylaminopropyl) carbodiimde hydrochloride    (EDC), 10.5 mL ethanolamine HCl Biacore (Catalog No. BR-1000-50)-   Biacore C Instrument with a PC compatible computer Biacore, (Catalog    No. BR-1100-51)-   Biacore C Control Software Biacore, as provided with Biacore C    instrument, version 1.0.1

Amine Coupling Kit BIA Certified: The kit contains one vial each: 115 mgNHS, 750 mg EDC, and 10.5 mL ethanolamine. Prepare each vial accordingto manufacturers directions. Aliquot 200 μL volumes of NHS and EDCsolutions into individual plastic/glass vials of appropriate size andcap. These solutions are stable for 2 months when stored at −20° C.Aliquot 200 μL of Ethanolamine into individual plastic/glass vials ofappropriate size and cap. This solution is stored at 2-8° C. and isstable according to manufacturer's directions.

To ensure good binding to the flow cell, a flow cell will be used forone week or 286 injections, which ever comes first. A new flow cell willbe immobilized at the beginning of each week. Immobilization of B7.1 Igin Preparation For Sample Testing. NOTE: Aliquot 200 μL of all solutionsinto 7 mm Biacore tubes for analysis. Thaw one vial containing B7.1 Igat ambient temperature. Dilute B7.1 Ig using 10 mM Acetate pH 5.0 buffer(1.7) to achieve a surface mass of between 3000-9000 Resonance Units(RU). Thaw one vial (200 μL) each of EDC, and NHS to ambienttemperature. Remove Ethanolamine HCl from the refrigerator and allowwarming to room temperature. From the Biacore software: Open thepublished project “B7 Ig Immobilization” selected from the“Immobilization Wizard” Open the published file “B7 Immob.blw.” Stepthrough the wizard and confirm or change selection by clicking “Next.”Under “User Information” select flow cell and provide experimentalinformation in the “Notebook” tab. Place reagent and ligand vials insample rack as outlined. Review instructions. Save the template file as:B7 Immob BIOQC# Date Initials Chip # Flow cell #.blw. Startimmobilization by clicking on “Start.” Save result file as: B7 ImmobBIOQC# Date Initial chip # flow cell # .blr. When the assay is finished,print the Wizard results and sensorgram.

Example 22 Carbohydrate Content Analysis of a CTLA4^(A29YL104E)-IgComposition, Tryptic Peptide Mapping and IEF

Tryptic Digest Peptide Mapping

In this trypsin digest method, CTLA4^(A29YL104E)-Ig samples aredenatured using guanidine-HCl, and reduced and alkylated using DTT andIAA. Samples are desalted using an NAP-5 column and digested withtrypsin. The digestion mixture is separated by reversed phase (C18)chromatography and peaks are detected by UV absorbance at 215 nm.

REAGENTS: Mobile Phase A solution (0.02% Trifluoroacetic Acid (TFA) inWater (v/v)); Mobile Phase B solution (0.02% TFA in 95% ACN(Acetonitrile) and 5% Water (v/v)); Alkylating Agent (200 mMIodoacetamide (IAA)); Dilution Buffer (100 mM Tris, 25 mM NaCl, pH 8.0);Denaturing Buffer (8 M Guanidine, 50 mM TRIS, pH 8.0); Digestion Buffer(50 mM TRIS, 10 mM CaC1₂, pH 8.0); Reducing Agent (100 mM DTT).

INSTRUMENTATION: (equivalent instrumentation may be used) NAP-5 columns(Amersham, cat. # 17-0853-02); HPLC Column Heater; Water's Alliance HPLCsystem with column heater and UV detector. An overview of this analysisis shown in FIG. 90.

Reduction and Alkylation: Samples (for example, CTLA4^(A29YL104E)-Ig,etc.) were diluted to 10 mg/ml by adding water to a final volume of 100μL (1 mg). 560 μL of denaturing buffer and 35 μL of Reducing Agent (100mM DTT) were added to the 100 μl samples, were mixed, and spun down in amicrocentrifuge for 3 seconds. Samples were then incubated at 50° C. for20 minutes ±2 minutes. 35 μL of Alkylating Agent (200 mM IAA) was thenadded to each sample, and again were mixed, and spun down in amicrocentrifuge for 3 seconds. Samples were then covered with aluminumfoil and incubated at 50° C. for 20 min. ±2 minutes. After the NAP-5columns were equilibrated by pouring 3 columns volumes (about 7-8 mL) ofdigestion buffer, 500 μl of the reduced and alkylated mixtures werepoured over the NAP-5 columns, allowing the liquid to drain throughcolumn. Samples were then collected from the NAP-5 columns via elutingsample off of the column with 1 mL of digestion buffer.

Digestion: Samples were digested with 20 μL of trypsin (0.5 μg/μL) in38° C. water bath for 4 hours (±0.5 hr). Upon completion of digest,samples were acidified with 2.5 μL of TFA. Samples were then placed intoautosampler vials for subsequent analysis.

Instrument Method: The instrument method is shown below:

Time (min) Flow (mL/min) Mobile Phase A Mobile Phase B 0 0.7 100 0 170.7 83 17 27 0.7 78 22 42 0.7 73 27 58 0.7 65 35 74 0.7 52 48 79 0.7 0100 84 0.7 100 0 88 0.7 100 0

The column was equilibrated with 100% Mobile Phase A buffer for 25minutes prior to the first injection. UV absorbance was monitored at 215nm while column temperature was manintained at 37° C. and theautosampler temperature at 4° C. A mobile phase A buffer blank was runbefore the first system suitability standard, thereafter followed by asingle 50 μL injection of each sample. A reference material injectionshould bracket every six sample injections.

Number of Theoretical Plates: Column efficiency, evaluated as the numberof theoretical plates, can be measured quantitatively using theretention time and the width of peak according to the Equation:

$N = {16\left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   “w” is the peak width at the baseline measured by extrapolating        the relatively straight sides to the baseline, “t” is the        retention time of the peak measured from time of injection to        time of elution of peak maximum.

If the N <50000, re-equilibrate the column.

Resolution: Determine The resolution (R) between 2 peaks, for examplepeak T2 and peak T12 as indicated in FIG. 31, can be determined usingthe following equation:

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{\left( {w_{1} + w_{2}} \right)}$

-   -   Where:    -   t₁, t₂=retention times of fragments peak T2 and peak T12,        respectively    -   w₁, w₂=tangent-defined peak width at baseline of the peaks with        retention times t₁ and t₂, respectively.

If R <1.5, the column should be re-equilibrate and if the problempersists, the column should be replaced.

FIG. 31 and Table 25 show the peptide fragments obtained from a trypsindigestion of CTLA4^(A29YL104E)-Ig. The region showing peptides T7 and T9at ˜50 minutes sometimes reflects incomplete sample digestion and peakscan show different qualities from day to day; however, within a run allsamples show comparability.

TABLE 25 Tryptic Peptide Fragments of CTLA4^(A29YL104E)-Ig FragmentResidue Theoretical Observed No. No. Mass Mass Peptide Sequence T1  1-14 1465.8 1464.8 MHVAQPAVVLASSR T2  15-28 1485.7 1484.8GIASFVCEYASPGK T3  29-33 666.3 666.4 YTEVR T4  34-38 586.7 586.4 VTVLRT5^(a)  39-83^(c) 4900.4 —^(d) QADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLR T6  84-93 1171.4 1170.5 AMDTGLYICK T7^(b)  94-128^(c)3983.5 —^(d) VELMYPPPYYEGIGNGTQIYVIDPEPCPD SDQEPK T8^(b) 129-132 435.4ND SSDK T9^(b) 133-158^(c) 3345.7 —^(d) THTSPPSPAPELLGGSSVFLFPPKPK T10159-165 834.9 834.5 DTLMISR T11 166-184 2140.3 2138.5TPEVTCVVVDVSHEDPEVK T12 185-198 1677.8 1677.8 FNWYVDGVEVHNAK T13 199-202500.6 500.3 TKPR T14^(a) 203-211^(c) 1189.2 —^(d) EEQYNSTYR T15 212-2271808.1 1808 VVSVLTVLHQDWLNGK T16 228-230 438.5 438.2 EYK T17 231-232307.4 ND CK T18 233-236 446.5 ND VSNK T19 237-244 838.0 837.4 ALPAPIEKT20 245-248 447.5 447.2 TISK T21 249-250 217.2 ND AK T22 251-254 456.5456.3 GQPR T23 255-265 1286.4 1285.6 EPQVYTLPPSR T24 266-270 604.7 604.3DELTK T25 271-280 1161.4 1160.7 NQVSLTCLVK T26 281-302 2544.7 2544GFYPSDIAVEWESNGQPENNYK T27 303-319 1874.1 1873.9 TTPPVLDSDGSFFLYSK T28320-324 574.7 574.3 LTVDK T29 325-326 261.28 ND SR T30 327-349 2803.092802.1 WQQGNVFSCSVMHEALHNHYTQK T31 350-356 659.7 659.2 SLSLSPG T6  84-931171.4 1170.5 AMDTGLYICK T7^(b)  94-128^(c) 3983.5 —^(d)VELMYPPPYYEGIGNGTQIYVIDPEPCPD SDQEPK T8^(b) 129-132 435.4 ND SSDK T9^(b)133-158^(c) 3345.7 —^(d) THTSPPSPAPELLGGSSVFLFPPKPK T10 159-165 834.9834.5 DTLMISR T11 166-184 2140.3 2138.5 TPEVTCVVVDVSHEDPEVK T12 185-1981677.8 1677.8 FNWYVDGVEVHNAK T13 199-202 500.6 500.3 TKPR T14^(a)203-211^(c) 1189.2 —^(d) EEQYNSTYR T15 212-227 1808.1 1808VVSVLTVLHQDWLNGK T16 228-230 438.5 438.2 EYK T17 231-232 307.4 ND CK T18233-236 446.5 ND VSNK T19 237-244 838.0 837.4 ALPAPIEK T20 245-248 447.5447.2 TISK T21 249-250 217.2 ND AK T22 251-254 456.5 456.3 GQPR T23255-265 1286.4 1285.6 EPQVYTLPPSR T24 266-270 604.7 604.3 DELTK T25271-280 1161.4 1160.7 NQVSLTCLVK T26 281-302 2544.7 2544GFYPSDIAVEWESNGQPENNYK T27 303-319 1874.1 1873.9 TTPPVLDSDGSFFLYSK T28320-324 574.7 574.3 LTVDK T29 325-326 261.28 ND SR T30 327-349 2803.092802.1 WQQGNVFSCSVMHEALHNHYTQK T31 350-356 659.7 659.2 SLSLSPG^(a)Peptides with N-linked carbohydrate ^(b)Peptides with O-linkedcarbohydrate ^(c)Masses for T5, T7, T9 and T14 are masses without thecarbohydrate moieties ^(d)A number of masses corresponding toglycosylated peptides were observed

Isoelectric Focusing

Isoelectric focusing (IEF) is used to evaluate the isoelectric points(pI) of the various isoforms of CTLA4^(A29YL104E)-Ig in both drugsubstance and drug product. This method uses Pharmacia BiotechAmpholine® PAGplates at pH gradient of 4.0-6.5 and a Multiphore IIFlatbed Electrophoresis System. Samples (for example,CTLA4^(A29YL104E)-Ig, etc.) are diluted in Milli-Q water and loadeddirectly onto the gel using sample application strips. The gel isfocused for 2.5 hours under increasing voltage using a 100 mM β-alaninesoaked cathode strip and a 100 mM glutamic acid/500 mM phosphoric acidsoaked anode strip. After focusing, the gel is fixed usingsulfosalicylic acid/trichloroacetic acid and then stained using aCoomassie blue staining system. After staining, the wet gel is scannedinto a digital image file using a laser-based densitometer at a 50 or100 μm spatial resolution with up to 4096 levels of optical densityresolution. CTLA4^(A29YL104E)-Ig focuses into 10 to 15 bands rangingfrom a pI of 4.5 to 5.5.

Isoelectric focusing of native CTLA4^(A29YL104E)-Ig on a gel (pH4.0-6.5) generates a similar banding pattern in the pI range of 4.6-5.5for the Process C Lot 224818-2004-007, Process B Lot 000929-287 andCo-mixture Lot 55128-162 as shown in FIG. 11. This procedure shows thatProcess B and C materials are comparable when analyzed on the same IEFgel.

Isoelectric focusing standards should be easily distinguished frombackground (See FIG. 11).

Protein Standard pI Lentil Lectin 8.65 8.45 Horse Myoglobin 7.35 6.85Conalbumin 5.90 Lactoglobulin 5.20 Soybean Trypsin Inhibitor 4.55Amyloglucosidase 3.50

CTLA4^(A29YL104E)-Ig is identified as multiple bands (>10) that have apI range from about 4.5 to about 5.5 (FIG. 31).

CTLA4^(A29YL104E)-Ig is a second generation CTLA4-Ig fusion glycoproteinwhich consists of the modified ligand-binding domain of cytotoxic Tlymphocyte antigen 4 (CTLA4) and the constant region of human IgG₁ heavychain. This novel molecule has therapeutic application as animmunosuppressant. CTLA4^(A29YL104E)-Ig contains multiple chargeisoforms which can be resolved by isoelectric focusing (IEF). An IEFmethod for the analysis of CTLA4^(A29YL104E)-Ig drug substance and drugproduct has been developed. This method is used to examineCTLA4A29YL104E-Ig in a AMPHOLINE® PAG plate pH 4.0-6.5 Multiphore IIflatbed electrophoresis system. CTLA4^(A29YL104E)-Ig drug substance,drug product, and reference material are diluted in Milli-Q water andloaded directly onto the gel. The gel is focused for 2.5 hours underincreasing voltage using a 100 mM β-alanine soaked cathode strip and a100 mM glutamic acid/500 mM phosphoric acid soaked anode strip. Afterfocusing, the gel is fixed and stained with Coomassie blue. The stainedgel is scanned by laser densitometry and semi-quantitative analysis ofgel bands is performed on the digital image file.

Materials:

Ampholine PAG Plate Gel GE Healthcare (Cat No. 80-1124-81) pH 4.0-6.5IEF Electrode Strips 6 × 280 mm GE Healthcare (Cat No. 80-1004-40)Sample Application pieces GE Healthcare (Cat No. 80-1129-46)

Equipment:

Multiphor II Electrophoresis GE Healthcare (Cat No. 18-1018-06) SystemCooling Plate 125 × 260 mm GE Healthcare (Cat No. 80-1106-54) PowerSupply NOVEX (Model Basic 3540) BioRad (Model PAC3000) ThermostaticCirculator VWR (Model 13271-074/ 1160S 1160A) Orbital Shaker IKA (ModelKS250/260) Personal Densitometer SI GE Healthcare (Model 375)ImageQuantTL Software GE Healthcare

Reagent Preparation:

Anode Buffer Solution (100 mL): 0.1 M Glutamic Acid in 0.5 M PhosphoricAcid; 3.4 mL 85% Phosphoric Acid ; 1.47±0.02 g Glutamic Acid; Milli-Qwater. Add Glutamic Acid to 50 mL of Milli-Q water. Add 85% phosphoricacid and Q.S. to 100 mL, stir to mix. Assign an expiration date of 6months and store at 4° C.

Cathode Buffer Solution (100 mL): 0.1 M β-Alanine, 0.9±0.02 g β-Alanine,Milli-Q water. Q.S. reagent to 100 mL with Milli-Q water, stir to mix.Assign an expiration date of 6 months and store at 4° C.

Fixing Solution (2000 mL): 3.5% 5-Sulfosalicylic Acid in 12%Trichloroacetic acid, 240±5.0 g Trichloroacetic Acid, 70±2.0 g5-Sulfosalicylic Acid, Milli-Q water. Combine reagents and Q.S. to 2000mL with Milli-Q water. Assign an expiration date of 3 months and storeat room temperature.

Apparatus and Gel Preparation. Connect the Multiphore II electrophoresisunit's cooling platform to the Multi-Temp thermostatic circulator andset the temperature to 10° C. Allow the circulator to reach 10±2° C.Remove the gel from the refrigerator. Using a scissors, carefully cutalong all four sides of the envelope making sure not to cut into thegel/gel support. Add approximately 1.0 mL of Milli-Q water to one edgeof the cooling platform. Place one edge of the gel/gel support into thewater so that the water moves across the entire edge of the gel.Carefully apply the gel across the cooling platform, avoiding theformation of air bubbles. Remove the transparent film from the surfaceof the gel. Soak each electrode strip with approximately 3.0 mL of theappropriate electrode solution (Table directly below). Apply theelectrode strips approximately 10 mm from the top and bottom edges ofthe gel. Place the cathode strip closest to the (−) marks and the anodestrip closest to the □□ (+) marks on the cooling platform. After theelectrode strips have been applied, cut the strips to fit the gel,avoiding contact with the gel support.

Electrode Solutions and Electrophoresis Parameter Settings pH AnodeCathode Voltage Current Power Time Range Solution Solution (V) (mA) (W)(h) 4.0-6.5 0.1M Glutamic 0.1M 2000 25 25 2.5 Acid in 0.5M β-AlanineH₃PO₄

Apply the sample application pieces approximately 10 mm above thecathode strip. Using the electrophoresis parameters defined in the Tabledirectly above, pre-focus the gel until the voltage reaches 300 V.

IEF pI Marker and Staining Control Preparation. Reconstitute the IEF pIMarker with 100 μL of Milli-Q water. Reconstitute the Carbonic AnhydraseII staining control with 1000 μL Milli-Q water to make a 1.0 mg/mL stocksolution. Add 10 μL of stock solution (1.0 mg/mL) to 90 μL Milli-Q waterfor a final loading concentration of 0.10 mg/mL.

Sample Preparation. Dilute the CTLA4^(A29YL104E)-Ig reference materialand samples to a concentration of 2 mg/mL. Example: If theCTLA4^(A29YL104E)-Ig sample has a concentration of 25 mg/mL, use thefollowing dilution to prepare the final loading concentration of 2mg/mL:

10 μL (of 25 mg/mL)+115 μL Milli-Q water=2 mg/mL

NOTE: If the sample concentration is 2.0 mg/mL, then load the samplewithout dilution.

Gel Loading. Load gels to facilitate sample identification based on therunning pattern. Do not load the gel symmetrically. Load the IEF pImarker, staining control, CTLA4^(A29YL104E)-Ig reference material, andCTLA4^(A29YL104E)-Ig samples as outlined in the Table directly below.Load all samples onto the sample application pieces.

Gel Loading Pattern

Loading Loading Protein Concentration Volume Load Lane Description(μg/μL) (μL) (μg) 1 IEF pI Marker* — 10.0 — 2 IEF pI Marker — 10.0 — 3CTLA4^(A29YL104E)-Ig 2.0 10.0 20 Reference Material 4 Sample 1 2.0 10.020 5 Staining Control 0.10 10.0 1.0 6 Sample 2 2.0 10.0 20 7CTLA4^(A29YL104E)-Ig 2.0 10.0 20 Reference Material 8 IEF pI Marker —10.0 — *IEF pI marker load in Lane 1 is necessary to define gelorientation. Begin the loading pattern within lane 2 and repeat theloading pattern for additional samples. The IEF pI marker must be loadedat least every tenth lane (Example: MRS₁S₂S₃S₄S₅S₆RM; M—marker;R—reference material, S_(x)—sample).

Gel Processing. Place the electrode holder onto the Multiphor II unitand align the electrodes with the center of the electrode strips on thegel. Connect the two electrodes from the electrode holder to the baseunit and place the safety lid in position. Using adhesive tape, coverthe holes in the safety lid to prevent the gel from drying. Connect theelectrodes to the power supply. Run the electrophoresis at theappropriate voltage, current, and power. When electrophoresis iscomplete, turn off the power supply and remove the safety cover andelectrode holder. Carefully remove the electrode strips and the sampleapplication pieces from the gel. Remove the entire gel and gel supportfrom the cooling plate and place in a 280×180×40 mm PYREX™ dishcontaining 200 mL fixing solution. Cover the dish with plastic wrap andplace on an orbital shaker at room temperature for a minimum of 20minutes. NOTE: The gel should be fixed for a maximum of 1 hour. Whenfixation is complete, wash the gel 3 times for 5 minutes each withapproximately 200 mL of Milli-Q water. Mix the GelCode Blue stainreagent solution by inverting the bottle several times. It is importantto mix the stain reagent before dispensing to ensure that a homogeneoussample of the reagent is used. Add approximately 200 mL of the stainreagent to the dish. Cover the dish with plastic wrap and place on anorbital shaker at room temperature for 18 to 20 hours to achieve optimalband development. When staining is complete, wash the gel by replacingthe stain reagent with approximately 200 mL Milli-Q water. Perform aminimum of 3 water changes over a 1-2 hour period for optimal results.

Gel Scanning and Analysis. Scan the gel using the scan parametersdefined in the Table directly above. Analysis of the gel is performed onthe scanned image file.

Gel Scanning and Analysis Parameters Setting Scan Parameters Scan PixelSize 100 Scan Digital Resolution 12 bits Band Detection ParametersMinimum Slope Initial 100 Noise Reduction Initial 10 % Maximum PeakInitial 0 Lane % width Set at 90% NOTE: Table 3 outlines generalguidelines for the analysis of gel images. Refer to the ImageQuant TL(v2003.03) manual and on-screen instructions for detailed information onthe appropriate adjustment of each band detection parameter.

Open a gel image file (scanned raw data) from <1D Gel Analysis> inImageQuantTL. Go to <Contrast> on toolbar and lower the <ImageHistogram> parameter until all bands are clearly visible. Select <LaneCreation> and choose <Manual> to set up <Number of Lanes> to beanalyzed. Adjust <Lane % Width> up to 100% to cover the gel lanes.Properly align single lanes if necessary. Use <Rolling Ball> method tosubtract background. This is not critical for IEF gel image analysis.Detect bands using the initial <Minimum Slope>, <Noise Reduction>, and<%Maximum Peak> settings listed in Table 3. Adjustment of these valuesis necessary to accurately identify bands. Manually correct any missedbands and misidentified bands. Compute band pI value by using thestandard pI marker from the labeled markers listed in the SystemSuitability Section for the pH/pI 4.0-6.5 gel. Do not perform thecalibration and normalization steps. Export the data contained withinthe Measurements Window into an Excel sheet for further calculation andreporting. Import the Excel data into the validated spreadsheet toperform quantitative analysis for reporting results.

SYSTEM SUITABILITY. Isoelectric focusing standards (pI markers) must bereadily distinguished from background and display limited distortion byvisual inspection of the scanned gel image (see Table directly below forthe listed pI markers).

Isoelectric focusing standards Protein pI Value Trypsinogen 9.30 Lentillectin, basic 8.65 Lentil lectin, middle 8.45 Lentil lectin, acidic 8.15Myoglobin, basic 7.35 Myoglobin, acidic 6.85 Carbonic anhydrase B(human) 6.55 Carbonic anhydrase B (bovine) 5.85 B-Lactoglobulin A 5.20Soybean Trypsin Inhibitor 4.55 Methyl red (dye) 3.75 Amyloglucosidase3.50 NOTE: Not all of the isoelectric focusing standards will appear onthe gel because the pH/pI range of the gel is 4.0-6.5. The pI markers at3.50, 4.55, 5.20, and 5.85 are to be identified and labeled on the gel.

The banding pattern of CTLA4^(A29YL104E)-Ig reference material and testarticles should display limited distortion by visual inspection of thescanned gel image. A staining control of carbonic anhydrase II (pI 5.4)at a low level of protein load (1.0 μg) is used to demonstrateconsistent gel staining. The band must be easily distinguished from thebackground by visual inspection of the scanned gel image.CTLA4^(A29YL104E)-Ig reference material must contain 8 to 15 bands withband intensity ≥1.0% within the pI range of 4.5 to 5.6.CTLA4^(A29YL104E)-Ig reference material bands within the pI range of 4.5to 5.6 must have a cumulative percent intensity of 95%.

DATA CALCULATION. The following equation is utilized for the calculationof the cumulative percent intensity of CTLA4^(A29YL104E)-Ig samplesrelative to reference material:

Cumulative Percent Intensity=Sample % Band Intensity (pI 4.5-5.6)×100

Reference % Band Intensity (pI 4.5-5.6)

Example: If the sample has a % Band Intensity (pI 4.5-5.6) of 95% andthe reference material has a % Band Intensity (pI 4.5-5.6) of 100%, theCumulative % Intensity will be 95%.

The CTLA4^(A29YL104E)-Ig material in one embodiment will have bands witha relative band intensity ≥1.0% within the pI range of 4.5-5.6. TheCTLA4^(A29YL104E)-Ig material has a cumulative percent intensityrelative to that of CTLA4^(A29YL104E)-Ig reference material within thepI range of 4.5-5.6.

Example 23 Transfection and Generation of Cell Lines

Prior to electroporation, the expression vector pD16 LEA29Y waslinearized with BstBI enzyme to produce compatible 4 bp overhangs. Thelinearized vector and sheared herring sperm carrier DNA (as carrier)were co-precipitated with ethanol and aseptically resuspended in PF CHOmedium (JRH Biosciences) for electroporation into DG44 cells.

Following electroporation, the cells were allowed to recover innon-selective medium. The cells were then seeded into 96 well plates inselective media of PF CHO containing 500 ng/mL of recombulin (Gibco), 4mM L-glutamine (Gibco) and methotrexate (ICN).

CTLA4^(A29YL104E)-Ig producing cell lines from this plating were chosenfor expression amplification using the following progression ofmethotrexate (MTX) concentrations added to the media:

20 nM⇒50 nM⇒100 nM⇒250nM⇒500nM⇒1 μM MTX.

Entire CTLA4^(A29YL104E)-Ig expression plasmid is integrated into thecell cell genome.

Production Cell Line Selection

The final production cell line GF1.1.9 was isolated after two rounds oflimiting dilution cloning of the best performing, amplified master wellcell lines. Selection of cell line GF1.1.9 was based on growth pattern,titer, and product containing a reduced amount of high molecular weightcomponent and higher sialic acid content relative to material producedfrom the other clones.

Example 24 Genetic Characterization of a CTLA4^(A29YL104E)-Ig

Genomic Stability Studies

DNA and RNA isolated from cells derived a cell bank were used forSouthern and Northern hybridization analysis, and sequencing of the cDNAfor a CTLA4^(A29YL104E)-Ig coding sequence. The results were comparedwith the results obtained from CTLA4^(A29YL104E)-Ig.

The results for the Northern hybridization analysis, and cDNA sequencingestimation are presented below.

Northern Hybridization Analysis

A culture inoculated with cells from the cell bank was expanded and usedto isolate RNA for the Northern hybridization analysis. The cultureprepared represents cells approximately 27 generations beyond the invitro cell age used in the CTLA4^(A29YL104E)-Ig production process.Total RNA was extracted from cells derived from theCTLA4^(A29YLL104E)-Ig cell bank and from cells from the expandedCTLA4^(A29YL104E)-Ig cells. A control utilizing total RNA from theparental CHO cell line was also used in these experiments. Approximately5 μg of total RNA was subjected to agarose gel electrophoresis underdenaturing conditions. The RNA in the gel was blotted onto a nylonmembrane and hybridized with a ³²P-labeled 1.2 kb HindIII/XbaI DNAfragment containing the CTLA4^(A29YL104E)-Ig gene. The 1.2 kgHindIII/XbaI DNA fragment used for the probe was isolated from plasmidpD16 LEA29Y.

A mRNA species of approximately 1.7 kilobases that hybridized to theCTLA4^(A29YL104E)-Ig gene probe was detected in the total RNA samplefrom the cell bank as shown in FIG. 32. Panel A and Panel B shown inFIG. 32 represent the ethidium bromide-stained agarose gel and thecorresponding autoradiogram, respectively.

These results indicate that only one transcript encodingCTLA4^(A29YL104E)-Ig is expressed in cultures derived from theCTLA4^(A29YL104E)-Ig expanded cell bank. In addition, no detectablechanges in the CTLA4^(A29YL104E)-Ig mRNA transcript were observed inthese samples as compared to the results obtained using the cell bank.

Example 25 Size Exclusion Chromatography

A size exclusion method has been developed to analyzeCTLA4^(A29YL104E)-Ig compositions using a 7.8 mm×300 mm TosoHaasTSK-3000 SWXL column equipped with a guard column with detection at 280nm. CTLA4^(A29YL104E)-Ig is evaluated for product homogeneity includingmonomer (single chain), dimer, or high molecular weight species (e.g.,tetramer). The method shows good precision (<2%) at a nominalconcentration of ˜10 mg/mL and is linear from ˜0.5-15 mg/mL (r²=0.999).The DL (Detection Limit) is ˜2.26 Φg/mL and the QL (Quantitation Limit)is ˜7.53 Φg/mL. These soluble CTLA4-Ig molecules are fusion proteinsconsisting of the ligand binding domain of cytotoxic T lymphocyteantigen 4 (CTLA4) and the constant region of human IgG1 heavy chain withpotential therapeutic application as immunosuppresants. These compoundsexert their physiological effects through binding to B7 antigens (CD80and CD86) on the surface of various antigen-presenting cells (APC), thusblocking the functional interaction of B7.1 and B7.2 with CD28 on thesurface of T-cells. This blockade results in the suppression of T-cellactivation, hence, the immune response. Although LEA29Y only differsfrom CTLA4Ig at two amino acid residues, Leu₁₀₄—glu and Ala₂₉—Try, themolecules have significantly different avidity towards B7.1 and B7.2antigens. LEA29Y shows a 5 to 10 fold greater avidity for the human formof B7.2 (CD86), and similar avidity for human B7.1 (CD80), compared withthe parental CTLA4Ig.

Size exclusion chromatography with a TSK-3000 SWXL column (7.8 mm×300mm) equipped with a guard column and detection at 280 nm is used toanalyze CTLA4^(A29YL104E)-Ig drug substance for homogeneity.CTLA4^(A29YL104E)-Ig dimer, high molecular weight (HMW) and lowmolecular weight (LMW) species are differentiated.

Size exclusion chromatography (SEC) is used to evaluateCTLA4^(A29YL104E)-Ig for product homogeneity. FIGS. 33A, 33B and 33Cshows the SEC chromatogram of CTLA4^(A29YL104E)-Ig for Process B,Process C and the co-mixture lot. SEC of CTLA4^(A29YL104E)-Ig indicatesthat the Process C material is 99.8 area percent dimer, 0.2 area percentHMW species and no detectable LMW species. These results are comparablewith the Process B material (dimer 97.4 area percent, HMW 2.6 areapercent, and LMW <DL). [001011] Reagents: 4N KOH (100 mL); SystemSuitability Standard (molecular wight markers dissolved in HPLC gradewater); Mobile phase Running Buffer (0.2 M KH₂PO₄, 0.9% NaCl, pH 6.8);4N NaOH; Dilution Buffer (25 mM NaH₂PO₄-H₂O, 10 mM NaCl, pH 7.5)

INSTRUMENTATION AND CONDITIONS—Equivalent instrumentation may besubstituted:

Pump Type Waters Model 600 Column Toso Haas 5 :m TSK 3000 SWXL, 300 mm ×7.8 m I.D. Hewlett Packard, (Catalog No. 79912S3-597) equipped with 5 :mTSK 3000 SWXL, 40 mm × 6.0 mm I.D. guard column, Hewlett Packard,(Catalog No. 79912S3-527) Detector Waters Model 486. Allow 15 minuteswarm up Wavelength 280 nm Flow Rate 1 mL/min Integration System VGMultichrom Injection System Waters Model 717 Plus Autosampler equippedwith refrigeration to 4° C. Injection Volume 20 mL Assay TargetConcentration 10 mg/mL Mobile Phase 0.2M KH₂PO₄, 0.9% NaCl, pH 6.8 withKOH Assay Run Time 20 min Column Temperature Ambient Retention TimeCTLA4^(A29YL104E)-Ig ~8.5 min ± 0.5 min, high molecular weight speciesat ~7.5 min ± 0.5 min

Standards and samples (10 mg/ml) were prepared as 50 ml volumes inlabeled autosampler vials. Samples were prepared in duplicate.

Calculations

Resolution (R) Determination and Retention Time Evaluation: 20 mL ofsystem suitability standards are injected to calculate the resolutionbetween 2 peaks from a chromatogram generated using such standards (forexample, one peak, Peak 1, having a retention time ˜8.5 minutes and asecond peak, Peak 2, having a retention time ˜10 minutes) using thefollowing equation:

${{Resolution}\mspace{14mu} (R)} = \frac{2\left( {t_{2} - t_{1}} \right)}{W_{2} + W_{1}}$

-   -   Where:    -   t₁=Retention time of Peak 1    -   t₂=Retention time of Peak 2    -   W₁=Peak width of Peak 1    -   W₂=Peak width of Peak 2

$\begin{matrix}{{{Resolution}\mspace{11mu} (R)} = {\frac{2\left( {t_{2} - t_{1}} \right)}{W_{2} + W_{1}} = \frac{2\left( {10.07 - 8.52} \right)}{{.57} + {.86}}}} \\{= {2.12{\exists\; 1.3}}}\end{matrix}$

Peak width equals width (in minutes) at the base of the peak afterextrapolating the relatively straight sides of the peak to the baseline.Retention time and peak widths are measured in the same units.

R must be ∃ 1.3 and the retention time for the peak should be ˜8.5 ∀ 0.5minutes.

Number of Theoretical Plates Determination: From the system suitabilitystandard chromatogram, the efficiency of the column can be determined bycalculating the number of theoretical plates according to the followingequation:

$N = {16\mspace{11mu} \left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   (t)=is the retention time of Peak 2 (in minutes)    -   (w)=is the width (in minutes) at baseline of Peak 2 obtained by        extrapolating the sides of the peak to the baseline as seen in        FIG. 1.

N should be ∃ 2500.

Integration of Peaks: The peak areas in the chromatogram were integrated(for example, FIGS. 33A, 33B and 33C). The CTLA4^(A29YL104E)-Ig dimerpeak is at ˜8.5 minutes and the high molecular weight species peak is at˜7.4 minutes.

Area percentages can be calculated according to the formulas below:

Area  %  Monomer = 100 − (Area  %  High  Molecular  Weight  Species + Area  %  Low  Molecular  Weight  Species)$\mspace{20mu} {{{Area}\mspace{14mu} \% \mspace{14mu} {High}\mspace{14mu} {Molecular}\mspace{14mu} {Weight}\mspace{14mu} {Species}} = {\frac{(B)}{(A) + (B) + (C)} \times 100}}$$\mspace{20mu} {{{Area}\mspace{14mu} \% \mspace{14mu} {Low}\mspace{14mu} {Molecular}\mspace{14mu} {Weight}\mspace{14mu} {Species}} = {\frac{(C)}{(A) + (B) + (C)} \times 100}}$

-   -   Where:    -   A=CTLA4^(A29YL104E)-Ig dimer peak area    -   B=Total area of all peaks with retention times less than        CTLA4^(A29YL104E)-Ig dimer    -   C=Total area of all peaks with retention times greater than        CTLA4^(A29YL104E)-Ig dimer peak (excluding inclusion volume).

[001022] The % RSD of the total area counts (excluding the inclusionvolume) is determined. The % RSD of the total area counts must be 2% orless.If area is <2707 area counts, report results as # DL, (DetectionLimit) (˜2.26 μg/mL). If area counts are between 2707-9014, reportresults as # QL (Quantitation Limit) (˜7.53 μg/mL). If area counts are9014, report results to nearest tenth of a percent.

Example 26 SDS-Page and Disulfide Bonds

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

A sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)procedure for the analysis of CTLA4^(A29YL104E)-Ig is used as a puritytest. Samples are prepared in a Tris-HCl (pH 6.8), SDS, sucrose, andbromophenol blue sample buffer in the presence (reduced) or absence(non-reduced) of dithiothreitol (DTT). Samples are placed in an 80° C.water bath for two minutes and electrophoresed in pre-cast, gradient(4-20%) polyacrylamide SDS gels using a Tris-glycine SDS running buffer.After electrophoresis, the gels are fixed and stained using a Coomassieblue or silver staining system. Non-reduced CTLA4^(A29YL104E)-Ig isobserved as one large band with an apparent approximate molecular weightof ˜10 μL kD. CTLA4^(A29YL104E)-Ig is observed as one major band with anapparent approximate molecular weight of ˜53 kD.

Samples of Process B Lot 000929-278, Process C Lot 224818-2004-007 andthe co-mixture Lot 551218-162 were electrophoretically resolved using4-20% gradient SDS-PAGE gels under both reducing and non-reducingconditions. Gels were separately stained with either Coomassie or silverstain as shown in FIG. 34 and FIG. 35. SDS-PAGE of non-reducedCTLA4^(A29YL104E)-Ig showed a major band at approximately 104 kDarepresenting the intact monomer. Three minor bands, not easily seen onelectronic reproductions, were also observed at ˜200, 65, and 53 kDa.Reduced samples of CTLA4^(A29YL104E)-Ig show a major band atapproximately 53 kDa representing the single chain form and a minor bandat ˜150 kDa. This comparison shows that the three tested materials arecomparable when analyzed on the same gel.

Disulfide Bonds

Disulfide bonds were characterized for Process C drug substance usingLot 224818-2004-007. Each chain of CTLA4^(A29YL104E)-Ig contains ninecysteines. These are Cys21, Cys48, Cys66, Cys92, Cys120, Cys171, Cys231,Cys277 and Cys335. Peptide mapping with on-line LC/MS/MS of both reducedand non-reduced CTLA4^(A29YL104E)-Ig was used to identify the sites ofintra-and intermolecular disulfide linkages in CTLA4^(A29YL104E)-Ig. Alist of the peptides obtained from peptide mapping of non-reducedCTLA4^(A29YL104E)-Ig along with the expected and observed MW is shown inTable 27.

The disappearance of certain peaks in the non-reduced peptide map andthe appearance of new peaks in the reduced peptide map provided evidenceof three disulfide-linked peptides: T2-T6, T11-T17, and T25-T30 whichcorresponded to the disulfide bonds of Cys21-Cys92, Cys171-Cys231 andCys277-Cys335. Peptides T5 and T7 have relatively high molecular weightand contain N-linked carbohydrates, which makes it difficult to locatedisulfide linkages. To generate shorter and carbohydrate-free peptides,CTLA4^(A29YL104E)-Ig was digested with a mixture of trypsin andchymotrypsin. As a result of the additional chymotrypsin cleavage, T7was shortened from a 35-amino-acid peptide to a 15-amino-acid peptide,designated T7′-T7′, in which the N-linked carbohydrate is removed.Disulfide-linked peptide T7′-T7′ appears in the unreduced map (see FIG.36). MS/MS on T7′-T7′ confirmed its sequence and inter-chain disulfidelinkage at Cys120-Cys120.

TABLE 27Peptide Sequence and MW of Disulfide-Linked Peptides from TrypsinDigestion of CTLA4^(A29YL104E)-Ig under Non-Reducing ConditionsDisulfide  Theoretical  Observed  Link Sequence MW MW T2-T6 (C21-C92)

2539.2 2539.6 T11-T17 (C171-C231)

2328.1 2328.4 T25-T30 (C277-C335)

3844.8 3846.3 T5 (C48-C66)

Glycopeptide^(a) T7-T7 (C120-C120)

Glycopeptide ^(a)Peptides T5 and T7-T7 give rise to several masses dueto heterogeneity of N-linked glycosylation making it difficult to locatedisulfide linkages.

Treatment with a mixture of trypsin and chymotrypsin also resulted inthe formation fragments, which corresponded to shorter versions of otherdisulfide linked peptides that were observed on hydrolysis ofCTLA4^(A29YL104E)-Ig by trypsin alone. These are shown in Table 28.

TABLE 28 Peptide Sequence and MW of Disulfide-Linked Peptides ofCTLA4^(A29YL104E)-Ig with (Trypsin and Chymotrypsin) DigestionDisulfide  Theoretical Observed  Link Sequence  MW MW T2′-T6′

872.4 872.4 T11-T17 (C171-C231)

2328.1 2328.6 T25′-T30′ (C277-C335)

984.5 984.5 T7′-T7′ (C120-C120)

3333.5 3333.2 T5 (C48-C66)

glycopeptide

The digestion of CTLA4^(A29YL104E)-Ig with a mixture of trypsin andchymotrypsin established the disulfide pairing in T7-T7 and confirmedthe disulfide bonds seen with digestion by trypsin alone. However, thisenzyme mixture did not have any effect on peptide T5, which is also aglycopeptide. In order to remove the N-linked carbohydrates from T5,CTLA4^(A29YL104E)-Ig was digested with a mixture of trypsin and elastaseas shown in FIG. 37. This mixture of enzymes hydrolyzed T5 at fourdifferent sites generating a shorter peptide designated (T5′-T5″) asshown in Table 29. This generated peptide had the expected mass of 1259Da and contained the disulfide linkage corresponding to Cys46-Cys66. Thepeptide map profile obtained from the hydrolysis of non-reducedCTLA4^(A29YL104E)-Ig by a mixture of trypsin and elastase, is shown inFIG. 37 and the sequence of peptide T5′-T5″ is in Table 29.

TABLE 29 Peptide Sequence and MW of Peptide T5′-T5″   Obtained by Digestion of CTLA4^(A29YL104E)-Ig    with a Mixture of Trypsin and Elastase Disulfide  Theoretical  Observed Link Sequence MW MW T5 (C48-C66)

Glycopeptide

The results indicate that CTLA4^(A29YL104E)-Ig has four intra-moleculardisulfide linkages at positions Cys21-Cys92 (T2-T6), Cys48-Cys66(corresponding to one single peptide T5), Cys171-Cys231 (T11-T17) andCys277-Cys335 (T25-T30) and one inter-chain disulfide linkage atpositions Cys120-Cys120 (T7-T7). The data accounted for all eighteencysteine residues. No mispairing was observed.

Example 27 CTLA4^(A29YL104E)-Ig Formulation

CTLA4^(A29YL104E)-Ig for Injection, 100 mg/vial is a sterilenon-pyrogenic lyophile. The composition of drug product is given inTable 30. It is a white to off white, whole or fragmented cake providedin Type I glass vials stoppered with gray butyl stoppers and sealed withaluminum seals. This product includes 10% overfill to account for vial,needle, and syringe holdup.

Prior to administration, CTLA4^(A29YL104E)-Ig for Injection, 100 mg/vialis constituted with 4.2 mL of Sterile Water for Injection, USP to yielda concentration of 25 mg/mL. It can be further diluted to aconcentration as low as 1 mg/mL with 5% Dextrose Injection, USP or 0.9%Sodium Chloride Injection, USP. Constituted and diluted solutions areclear, colorless and essentially free of particulate matter on visualinspection.

TABLE 30 Composition of CTLA4^(A29YL104E)-Ig for Injection, 100 mg/vialQuantity per Vial Component Function (mg) CTLA4^(A29YL104E)-Ig ActiveIngredient 110^(a) Sucrose Lyoprotectant 220 Sodium Phosphate MonobasicBuffering Agent  15.18 Monohydrate Sodium Chloride Ionic Strength  2.55Adjustment 1N Sodium Hydroxide pH Adjustment To 7.5 ± 2 1N HydrochloricAcid pH Adjustment To 7.5 ± 2 Water for Injection^(b) Solvent q.s. to5.5 mL ^(a)Each vial contains 10% overfill for vial, needle and syringeholdup of the reconstituted solution. ^(b)Removed during lyophilization

The glass transition temperature of the frozen solution to be freezedried was determined to be −28.9° C. Freeze drying studies wereconducted at various shelf temperatures in order to determine thehighest possible shelf temperature allowable during primary drying,without compromising product quality. Based on these studies, a shelftemperature of −20° C. was selected for the primary drying step duringthe freeze-drying of CTLA4^(A29YL104E)-Ig. At the end of thefreeze-drying cycle, the vials are stoppered under reduced pressure.

The production process involves freezing vials containing bulk solutionfor lyophilization (with appropriate excipients) in a freeze dryerchamber, followed by sublimation of frozen water under controlledtemperature and pressure. Temperature and pressure conditions in thechamber are optimized in order to have efficient sublimation withoutcompromising product quality.

Compatibility of the solution with various product contact surfaces andpackaging components was studied. The solution was found to becompatible with stainless steel 316 L, silicone tubing, Acrodisc™, HTTuffryn (polysulfone), Millipore PVDF (polyvinylene fluoride) filtermembranes and the selected container closure system.

CTLA4^(A29YL104E)-Ig for Injection, 100 mg/vial is packaged in 15 ccType I flint tubing glass vials and stoppered with a 20 mm Daikyo graybutyl D-21-7-S/B2-TR fluro-resin coated stopper and sealed with a 20 mmaluminum flip-off seal.

Vial selection for CTLA4^(A29YL104E)-Ig for Injection was based on thefill volume of 5.5 mL to ensure efficient freeze-drying and 20 mm Daikyogray butyl D-21-7-S/B2-TR fluro-resin coated stopper selection was basedon the compatibility data.

Extensive use time compatibility studies have been conducted.CTLA4^(A29YL104E)-Ig for Injection 100 mg/mL when constituted to 25mg/mL with sterile water for injection may be stored at ambient roomtemperatures from 15°-25° C. (59°-77° F.) and room light for 24 hours.Constituted solution when further diluted to either 1 mg/ml or 10 mg/mLwith 0.9% Sodium Chloride Injection (Normal Saline/NS) or with 5%Dextrose Injection (D5W) and stored in either a PVC or Intra Via non-PVCbag at ambient room temperatures from 15-25° C. (59°-77° F.) and roomlight, showed no loss in potency or increase in high molecular weightspecies over a period of 24 hours. The diluted solution must be filteredthrough a 0.2 μm or a 1.2 μm mixed cellulose/acetate filter prior toadministration. The diluted solution is compatible with 0.2 μm and 1.2μm mixed cellulose/acetate filters.

The product is incompatible with silicone. It interacts with silicone toform visible particles. Therefore, contact with silicone treatedsurfaces such as siliconized syringes should be avoided.

Example 28 CTLA4-Ig Production Process

CTLA4-Ig is produced as a secreted protein in large-scale cell cultureusing a Chinese hamster ovary (CHO) cell line. The CTLA4-Ig productionprocess is initiated using a series of flask and seed bioreactorinoculum expansion steps. The contents of a final seed bioreactor areused to inoculate a 5000-L production bioreactor. The cell cultureharvest from the 5000-L production bioreactor is clarified andconcentrated by microfiltration and ultrafiltration. The cell-freeharvest material is adjusted for pH and conductivity in preparation fordownstream processing. CTLA4-Ig is purified using a series ofchromatographic and filtration steps. The downstream CTLA4-Ig productionprocess includes two anion exchange chromatography steps, onehydrophobic interaction chromatography step and one affinitychromatography step. The purpose of these steps is to purify theCTLA4-Ig protein, to remove high molecular weight CTLA4-Ig material andto control the sialic acid content of the CTLA4-Ig drug substance. Thedownstream processing steps also include a viral inactivation step and aviral filtration step to clear potential adventitious viral agents.Purified CTLA4-Ig drug substance is filled into 2-L polycarbonatebottles and frozen at a target temperature of −70° C. prior to storageat a target temperature of −40° C. The frozen drug substance is thawed.

N-acetylneuraminic acid (NANA) is the primary sialic acid speciespresent in CTLA4-Ig drug substance. References to sialic acid throughoutthis section refer specifically to this species. Minor levels ofN-glycolylneuraminic acid (NGNA) are also present in CTLA4-Ig drugsubstance. The levels of both NANA and NGNA are determined for the finalCTLA4-Ig drug substance. A process flow diagram for the CTLA4-Igproduction process is shown in FIG. 91.

CTLA4-Ig is produced in 5000-L production bioreactors with anapproximate working volume of 4300 L. One batch of CTLA4-Ig drugsubstance is made from a single production bioreactor derived from asingle vial from the cell bank. The upstream cell culture productionprocess is initiated using a single vial of cells from a cell bank. Thevial is thawed and the entire contents used to seed a T-flask containingcell culture growth medium. The cells are then expanded in a series ofspinner flasks. Flasks from the final spinner flask inoculum expansionstep are used to inoculate the 140-L seed bioreactor. The 140-L seedbioreactor has a working volume of approximately 100 L. The contents ofthe 140-L seed bioreactor are used to inoculate the 1100-L seedbioreactor. The 1100-L seed bioreactor has a working volume ofapproximately 600 L. The contents of the 1100-L seed bioreactor are usedto seed the 5000-L production bioreactor. The cells are cultivated inthe 5000-L production bioreactor for approximately 14 days. Followingthe production bioreactor step, the cell culture broth from a singlebioreactor is transferred to a harvest vessel for further processing. Atangential flow microfiltration (MF) unit is used to separate thesecreted CTLA4-Ig protein from host cells and cell debris. The CTLA4-Igprotein-containing MF permeate is then concentrated by ultrafiltration(UF) and adjusted for pH and conductivity in preparation for the firstchromatography step.

The purification and downstream processing steps for CTLA4-Ig drugsubstance consist of anion exchange chromatography, hydrophobicinteraction chromatography (HIC), viral inactivation, affinitychromatography, tangential flow UF concentration and diafiltration,viral filtration, a second anion exchange chromatography and UFconcentration and diafiltration. The HIC step utilizes multiple cyclesper CTLA4-Ig batch depending on the quantity of CTLA4-Ig to beprocessed. In-process material from multiple HIC step cycles are pooledfor the subsequent viral inactivation step. In-process material fromdifferent lots are not pooled. Multiple viral filters may be used inparallel to process a single lot of CTLA4-Ig. Following viralfiltration, the filtrates are pooled for further processing.

Each lot of CTLA4-Ig is filtered through a 0.2 μm filter into 2-Lpolycarbonate (PC) bottles and temporarily stored at 2° to 8° C. The 2-LPC bottles of CTLA4-Ig are frozen at a target temperature of -70° C. andthen stored at a target temperature of −40° C. Bottles of drug substanceare thawed in an incubator at 22° to 24° C. and cooled to 2° to 8° C.prior to shipment.

Production

CTLA4-Ig is produced in large-scale cell culture using a Chinese hamsterovary (CHO) cell line. The CTLA4-Ig upstream production process isinitiated with the thaw of a frozen vial from a cell bank. The cultureis propagated in a T-flask, followed by a series of spinner flaskcultures. These cultures are transferred to a 140-L seed bioreactor. Theculture from the 140-L seed bioreactor is transferred to a 1100-L seedbioreactor. The culture from the 1100-L seed bioreactor is used toinoculate a 5000-L production bioreactor. The production bioreactor isharvested primarily based on a target sialic acid to CTLA4-Ig proteinmolar ratio. The cell culture harvest is clarified and concentratedusing a combination of microfiltration (MF) and ultrafiltration (UF).Finally, the concentrated cell-free harvest material is adjusted toachieve a specified conductivity and pH in preparation for downstreamprocessing.

Cell Culture and Feed Media Preparation

Solid and liquid media components are weighed and measured. Two cellculture media are used in the process. Medium 127-G is used in theT-flask, spinner flask, seed bioreactor and production bioreactor steps.Medium 117-E is used as a feed medium in the production bioreactor step.The composition of Medium 127-G is shown in the table directly below.

Component Concentration CD-CHO 25x Acid Solubles I 40.0 mL/L CD-CHO 25xAcid Solubles II 40.0 mL/L CD-CHO 25x Salts I 40.0 mL/L CD-CHO 25x SaltsII 40.0 mL/L L-Glutamine 0.585 g/L r-human Insulin 0.1 mL/L (10 mg/mLsolution) Methotrexate (20 mM solution) 5 μL/L Sodium Bicarbonate 2.22g/L Water For Injection As required 1N HCl Solution 0-5 mL/L to adjustpH 10N NaOH Solution 0-10 mL/L to adjust pH

The composition of Medium 117-E is shown below.

Component Concentration eRDF-1 Medium 16.47 g/kg Dextrose 30.29 g/kgD-Galactose 12.38 g/kg L-Glutamine 4.02 g/kg r-human Insulin (10 mg/mLsolution) 0.98 mL/kg TC Yeastolate 4.90 g/kg Water For Injection Asrequired 1N HCl Solution 0-5 mL/kg to adjust pH 10N NaOH Solution 0-2mL/kg to adjust pH

The composition of e-RDF-1 Medium is below:

Component Concentration (mg/L) Cupric Sulfate 5 H₂O 0.0008 FerrousSulfate 7 H₂O 0.220 Magnesium Sulfate (MgSO₄) 66.20 Zinc Sulfate 7 H₂O0.230 Sodium Pyruvate 110.0 DL-Lipoic Acid Thioctic 0.050 Linoleic Acid0.021 L-Alanine 6.68 L-Arginine 581.44 L-Asparagine 94.59 L-AsparticAcid 39.93 L-Cystine 2 HCl 105.38 L-Glutamic Acid 39.7 Glycine 42.8L-Histidine HCl—H₂O 75.47 L-Isoleucine 157.40 L-Leucine 165.30 L-LysineHCl 197.26 L-Methionine 49.24 L-Phenylalanine 74.30 L-Proline 55.3L-Hydroxyproline 31.5 L-Serine 85.10 L-Threonine 110.8 L-Tryptophan18.40 L-Tryosine 2 Na 2H₂O 108.10 L-Valine 108.9 Para Amino Benzoic Acid0.51 Vitamin B12 0.339 Biotin 1.00 D-Ca Pantothenate 1.29

The table is continued below:

Component Concentration (mg/L) Choline Chloride 12.29 Folic Acid 1.96i-Inositol 46.84 Niacinamide 1.47 Pyridoxal HCl 1.00 Pyridoxine HCl0.420 Riboflavin 0.21 Thiamine HCl 1.59 Putrescine 2HCl 0.020

The 127-G cell culture medium used in the T-flask and spinner flasks inthe process is prepared in medium vessels equipped with an agitator formixing and a graduated sight glass for volume determination. The batchsize of Medium 127-G used in the T-flask and spinner flask inoculumexpansion steps is 75 L. Medium 127-G is prepared using Water ForInjection (WFI). Solid and liquid medium components are added to theWFI. The medium is mixed for the required period of time after theaddition of each component. WFI is added to bring the medium to thefinal batch volume of 75 L. A sample is removed from the final mediumpreparation and the glucose concentration, pH, and osmolality of thesample are measured to ensure that the medium meets the definedacceptance criteria. The medium is filtered through a 0.2 μm filter anddispensed into sterile polyethylene terephthalate glycol (PETG) bottles.The Medium 127-G prepared for the T-flask and spinner flask inoculumexpansion steps is stored at 2° to 8° C. for a maximum of 42 days.Medium 127-G for the 140-L and 1100-L seed bioreactor steps is preparedin vessels equipped with an agitator for mixing. The batch size ofMedium 127-G used in the 140-L seed bioreactor step is 120 L. The vesselused to prepare the medium for the 140-L seed bioreactor is equippedwith a graduated sight glass for volume determination. The batch size ofMedium 127-G used in the 1100-L seed bioreactor step is 600 kg. Thevessel used to prepare the medium for the 1100-L seed bioreactor isequipped with a differential pressure transmitter for weightdetermination.

The required volumes of Medium 127-G are transferred to the 140-L and1100-L seed bioreactors through consecutive 0.2 ocm and 0.1 μm filters.The Medium 127-G prepared for the 140-L and 1100-L seed bioreactor stepsmay be held at 37° C. for a maximum of 48 hours. The medium may be heldat 4° C. for an additional 84 hours.

Preparation of 127-G and 117-E Cell Culture Media Used in a 5000-LProduction Bioreactor Step.

Medium 127-G for the 5000-L production bioreactor step is prepared in amedium preparation vessel equipped with an agitator and differentialpressure transmitter for weight determination. The batch size of Medium127-G used in the 5000-L production bioreactor step is 2900 kg. Therequired volume of Medium 127-G is transferred to the 5000-L productionbioreactor through consecutive 0.2 μm and 0.1 μm filters. The Medium127-G prepared for the 5000-L production bioreactor step may be held at37° C. for a maximum of 48 hours. The medium may be held at 4° C. for anadditional 84 hours.

Feed medium 117-E is prepared in a medium preparation vessel equippedwith an agitator for mixing and a differential pressure transmitter forweight determination. The batch size of Medium 117-E used in the 5000-Lproduction bioreactor step is 1800 kg. The Medium 117-E components areadded to a specified weight of WFI in the medium preparation vessel. Themedium is mixed for the required period of time after the addition ofeach component. WFI is added to bring the medium to the specified finalweight. A sample is removed from the final medium preparation and theglucose concentration, pH, and osmolality of the sample measured inorder to ensure that the medium meets the defined acceptance criteria.The required volume of Medium 117-E is transferred to a feed mediumholding tank through consecutive 0.2 μm and 0.1 ∝m filters. The Medium117-E prepared for the 5000-L production bioreactor step may be held at37° C. for a maximum of 2 days. The medium may be held at 4° C. for anadditional 4 days.

Inoculum Expansion Steps in the T-Flask and Spinner Flask InoculumExpansion Steps

The objective of the T-flask and spinner flask inoculum expansion stepsof the CTLA4-Ig production process is to serially propagate cells fromthe cell bank vial to provide a sufficient number of viable cells toinoculate the 140-L seed bioreactor. A single vial from the cell bank isremoved from the vapor phase of a liquid nitrogen storage freezer andthawed in a water bath at 37° C. The entire contents of the vial areaseptically transferred into a sterile 15-mL conical centrifuge tube.Medium 127-G is added to bring the final volume to 10 mL. The cellsuspension is centrifuged, the supernatant discarded and the cell pelletresuspended in 10 mL of 127-G cell culture medium. The resuspended cellsare transferred to a T-175 flask containing 10 mL of Medium 127-G. Theviable cell density and the percent viability of the culture in theT-175 flask are determined. The percent viability at this step of ≥84%was established. Medium 127-G is added to the T-175 flask to achieve atarget viable cell density of 2.1×10⁵ cells/mL.

The T-175 flask is incubated at 37° C. in an atmosphere of 6% carbondioxide for a maximum of four days to achieve a target final cell numberof 1.80×10⁷ viable cells. Following the T-175 flask step, the culture isexpanded using a series of 0.25-L, 1-L, and 3-L spinner flask steps. Ateach passage, the cells are seeded at a target density of 2.0×10⁵ viablecells/mL. The spinner flask cultures are incubated at 37° C. in anatmosphere of 6% carbon dioxide.

Cell culture material from the final 3-L spinner flask inoculumexpansion step is pooled in a sterilized 20-L inoculum transfer vessel.The final viable cell density at the 3-L spinner flask inoculumexpansion step of 1.0 to 2.0×10⁶ cells/mL and a minimum percent cellviability of ≥80% were established. These exemplary values ensure that asufficient number of viable cells is used to inoculate the 140-L seedbioreactor. A total volume of 12 L to 18 L of the pooled cell culturefrom the final 3-L spinner flask inoculum expansion step is used toinoculate the 140-L seed bioreactor.

140-L and 1100-L Seed Bioreactor Inoculum Expansion Steps

The objective of the 140-L and 1100-L seed bioreactor inoculum expansionsteps of the CTLA4-Ig production process is to provide a sufficientnumber of viable cells to inoculate the 5000-L production bioreactor.The seed bioreactors are operated in batch mode using cell culturemedium 127-G. Temperature, pH, dissolved oxygen, pressure, agitation andgas flow rates for air, oxygen, and carbon dioxide are controlled by adistributed control system (DCS) and provide conditions for optimalgrowth of the culture in the seed bioreactors. The seed bioreactors areoperated at 37° C. Culture samples are removed from the seed bioreactorsfor the determination of viable cell density, percent viability andmetabolite concentrations.

The 140-L seed bioreactor is inoculated with pooled inoculum from the3-L spinner flask inoculum expansion step to a target initial viablecell density of 2.0×10⁵ cells/mL. The final viable cell density at the1100-L seed bioreactor inoculum expansion step of 1.0 to 2.5×10⁶cells/mL and a minimum percent cell viability of ≥80% were established.These acceptance criteria ensure that a sufficient number of viablecells is used to inoculate the 5000-L production bioreactor. The cellculture from the 1100-L seed bioreactor is transferred to the 5000-Lproduction bioreactor to achieve a target initial viable cell density of1.5×10⁵ cells/mL.

Production Bioreactor Step

The objective of the 5000-L production bioreactor step is to expand thenumber of viable cells and to produce the CTLA4-Ig protein. The durationof the production bioreactor step is approximately 14 days. Inoculumfrom the 1100-L seed bioreactor is seeded into a 5000-L productionbioreactor containing cell culture medium 127-G. The productionbioreactor is operated in fed-batch mode. Temperature, pH, dissolvedoxygen, pressure, agitation and gas flow rates for air, oxygen, andcarbon dioxide are controlled by the DCS and provide conditions foroptimal growth of the culture and production of the CTLA4-Ig protein inthe production bioreactor.

A three-stage temperature control strategy is used during the 5000-Lproduction bioreactor step to optimize cell growth and CTLA4-Igproduction. The initial incubation temperature of the productionbioreactor is controlled at 37° C. to achieve optimal cell growth. Thetemperature is lowered to 34° C. when a viable cell density of 4.0×10⁶cells/mL is achieved in the production bioreactor or at 144 hours fromthe time of inoculation, whichever occurs first. The temperature islowered to 32° C. at 240 hours and maintained at 32° C. until harvest.Daily samples are obtained from the 5000-L production bioreactor tomonitor cell growth, cell viability, metabolite concentrations, CTLA4-Igtiter and the sialic acid to CTLA4-Ig protein molar ratio.

Feeding of Medium 117-E to the production bioreactor is initiatedbetween 12 to 24 hours from the time of inoculation. Medium 117-E isadded daily to achieve a target of 1% (v/v) of feed medium to culturevolume or a sufficient volume of the feed medium 117-E to bring theglucose concentration to 3 g/L. This feeding strategy providessufficient levels of glucose and other nutrients to the culture tosupport the production of CTLA4-Ig protein during the productionbioreactor step.

Medium 117-E is supplemented with D-galactose to promote increasedglycosylation of CTLA4-Ig protein. Galactose supplementation results inan increase in the terminal sialic acid content of the CTLA4-Ig protein.The sialic acid to CTLA4-Ig protein molar ratio is an important harvestcriterion in the CTLA4-Ig production process.

A three-stage strategy is also used to control the dissolved oxygen andagitation rate in the production bioreactor. The initial agitation rateof 30 rpm ensures uniformity of physical conditions and preventssettling of the cells within the 5000-L production bioreactor. Theinitial dissolved oxygen setpoint of 40% ensures availability ofsufficient levels of dissolved oxygen to support the growth of theculture in the production bioreactor. The setpoints for dissolved oxygenand agitation rate are increased at 96 hours from the time ofinoculation to 50% and 40 rpm, respectively. At 120 hours from the timeof inoculation, the setpoints for dissolved oxygen and agitation rateare further increased to 60% and 50 rpm, respectively. This strategyensures sufficient levels of dissolved oxygen to maintain the cellculture during the production bioreactor step. The titer of CTLA4-Igprotein increases during the course of the production bioreactor step.The culture viability is monitored throughout the course of this step.The sialic acid to CTLA4-Ig protein molar ratio is monitored twice dailyfrom 6 days from the time of inoculation until the time of harvest. Thesialic acid to CTLA4-Ig protein molar ratio peaks at approximately 10 ataround day 8 from the time of inoculation and then decreases graduallyover the remainder of the production bioreactor step. The primaryharvest criterion for the production bioreactor is the sialic acid toCTLA4-Ig protein molar ratio. The production bioreactor is harvested ata target sialic acid to CTLA4-Ig protein molar ratio of 8.0.

A minimum cell viability value of 38% was also established for harvestof the culture. These harvest criteria ensure the consistency of thein-process harvest material for downstream processing to CTLA4-Ig drugsubstance. The total number of cell generations from the initiation ofthe inoculum expansion through the harvest of the production bioreactorin the CTLA4-Ig upstream production process is approximately 38generations. The cell line used in the process was demonstrated to bestable for 105 generations in a cell line stability study.

Harvest Operation Steps

The objective of the harvest operation steps is to remove cells and celldebris from the harvest material and to concentrate the in-processstream containing CTLA4-Ig protein for further downstream processing.The MF and UF systems are sanitized prior to processing the harvestmaterial. The MF and UF systems are flushed with a peracetic acidsolution. The MF and UF systems are then treated with a bleach solutionand a sodium hydroxide solution, respectively. Finally, the MF and UFsystems are flushed with WFI until a conductivity of ≤3 μS/cm in theretentate and permeate is achieved. The cell culture broth from the5000-L production bioreactor is transferred to a harvest vessel.Tangential flow MF with 0.65 μm polyvinylidene fluoride membranes isused for the removal of cells and cell debris from the in-processharvest material containing CTLA4-Ig protein. The cell-free MF permeateis collected in a permeate vessel. The cell-free MF permeate issimultaneously concentrated by tangential flow UF using polyethersulfonemembranes of 30 kilodalton (kDa) nominal molecular weight cutoff. The UFpermeate is used as the diafiltration medium for the MF process step. A0.1 N phosphoric acid solution is used for storage of the MF system. A0.1 N sodium hydroxide solution is used for storage of the UF system.Temperature, permeate and retentate flowrates and transmembranepressures are monitored and controlled during the MF and UF operation.Flow rates are measured by in-line flowmeters present on the filtrationskids. Sensors are used to measure pressure and temperature. The MFretentate flow rate of 163 to 235 L/min and the MF transmembranepressure of ≤3.8 psig were established. These values ensure consistencyin the performance of the harvest operation steps.

The final step in the harvest operation is a pH and conductivityadjustment of the clarified and concentrated in-process harvestmaterial. The pH and conductivity of the concentrated in-process harvestmaterial are adjusted for capture of the CTLA4-Ig protein during thefirst downstream chromatography processing step. The pH is adjusted to8.0 by the addition of a 2 M Tris solution and the conductivity of theconcentrated permeate is reduced to 10 mS/cm by the addition of WFI. Theconcentrated and adjusted in-process harvest material is then filteredthrough three parallel and one consecutive filter housing containing 0.2μm disposable filters and transferred to a surge tank in the downstreampurification area.

TABLE 31 Composition of CD-CHO Medium Component Concentration CD-CHO 25xAcid Solubles I 40.0 mL/L CD-CHO 25x Acid Solubles II 40.0 mL/L CD-CHO25x Concentrate Salt I 40.0 mL/L CD-CHO 25x Concentrate Salt II 40.0mL/L L-Glutamine 0.585 g/L r-human Insulin (10 mg/mL Solution) 0.1 mL/LMethotrexate (25 mg/mL Solution) 0.0018 mL/L Sodium Bicarbonate 2.22 g/LWater For Injection As required 1M HCl Solution As required to adjust pH10N NaOH Solution As required to adjust pH

TABLE 32 Composition of eRDF Feed Medium Component Concentration eRDF-1Medium 16.80 g/L Dextrose 30.9 g/L D-Galactose 12.6 g/L L-Glutamine 4.10g/L r-human Insulin (10 mg/mL Solution) 1.00 mL/L TC Yeastolate 5 g/LWater For Injection As required 1M HCl Solution As required to adjust pH10N NaOH Solution As required to adjust pH

TABLE 33 Composition of Modified 50% CD-CHO Medium ComponentConcentration CD-CHO 25x Acid Solubles I 20.0 mL/L CD-CHO 25x AcidSolubles II 20.0 mL/L CD-CHO 25x Concentrate Salt I 20.0 mL/L CD-CHO 25xConcentrate Salt II 20.0 mL/L D-Galactose 0.4 g/L r-human Insulin (10mg/mL Solution) 0.05 mL/L Sodium Bicarbonate 1.11 g/L Water ForInjection As required 1M HCl Solution As required to adjust pH 10N NaOHSolution As required to adjust pH

Example 29 CTLA4-Ig Purification Process

The final pH-and conductivity-adjusted material from the harvestoperation step described in Example 28 is first processed using an anionexchange chromatography step. The product pool from this first anionexchange chromatography step is then processed using a hydrophobicinteraction chromatography step. The CTLA4-Ig-containing material isthen treated with Triton X-100 to inactivate potential adventitiousviral agents. The Triton X-100-treated material is processed using arecombinant Protein A affinity chromatography step. The product poolfrom the recombinant Protein A chromatography step is concentrated anddiafiltered. A viral filtration step for the removal of potentialadventitious viral agents is then performed. The filtrate is furtherpurified using a second anion exchange chromatography step. Finally, thepurified CTLA4-Ig protein is concentrated and diafiltered into the finaldrug substance buffer.

CTLA4-Ig is a genetically-engineered fusion protein which consists ofthe functional binding domain of human Cytotoxic T-Lymphocyte Antigen-4and the Fc domain of human monoclonal immunoglobulin of the IgG1 class.CTLA4-Ig dimer is comprised of two homologous glycosylated polypeptidechains of approximately 46 kDa each which are covalently linked througha single disulfide bond. A process flow diagram for the downstream stepsof CTLA4-Ig production process is shown in FIG. 92. CTLA4-Ig is producedin 5000-L production bioreactors using a Chinese hamster ovary (CHO)cell line. Chromatographic and filtration steps in the downstreamCTLA4-Ig production process are performed at ambient temperature.In-process material is stored at 2 to 8° C. between processing steps.The downstream process is initiated with the receipt of in-processharvest material from the harvest operation steps. This material isfirst processed through an anion exchange chromatography column using QSepharose™ Extreme Load (QXL) resin. The QXL column functions to capturethe CTLA4-Ig protein from the harvest material. The QXL column capturestep also accomplishes a volume reduction for further downstreamprocessing.

The QXL product pool is passed through a hydrophobic interactionchromatography (HIC) column utilizing a Phenyl Sepharose Fast Flowresin. During this step, CLTA4-Ig high molecular weight (HMW) materialand Chinese hamster ovary host cell proteins (CHOP) are bound to thecolumn. The CLTA4-Ig dimer protein does not bind to the HIC resin andpasses through the column. Following the HIC step, a viral inactivation(VI) using Triton® X-100 detergent is performed to inactivate potentialadventitious viral agents. The next step utilizes a recombinant ProteinA Sepharose Fast Flow (rPA) resin affinity column. In thischromatography step, the levels of CHOP and monocyte chemotactic protein1 (MCP-1) are reduced. The rPA step is defined as a viral clearancestep. Following affinity chromatography step, the CLTA4-Ig protein isconcentrated and dialyzed using 30 kDa ultrafiltration (UF) membranesand processed through PlanovaTM 15 nm filters to remove potentialadventitious agents. Upon completion of the viral filtration (VF) step,the CLTA4-Ig protein is processed on a second anion exchange columnusing Q Sepharose Fast Flow (QFF) resin. The QFF chromatography stepreduces residual recombinant protein A and DNA levels. The QFF step isdefined as a viral clearance step. Finally, the CLTA4-Ig protein isconcentrated and diafiltered using a 30 kDa UF membrane against asolution of 25 mM sodium phosphate, 50 mM NaCl, pH 7.5. The CLTA4-Igdrug substance is then filtered through a 0.2 μm filter prior to thefinal fill step.

The process-related impurities CHOP, MCP-1, residual recombinant proteinA, DNA and Triton X-100 are reduced at specific downstream processingsteps of the CTLA4-Ig production process. PPs with action limits areestablished at the primary in-process control point for each impurity.₃Product pool CHOP and MCP-1 are identified as PPs for the rPAchromatography step. Product pool residual recombinant protein A ligand,DNA and Triton X-100 are identified as PPs for the QFF chromatographystep. Based on the known structure and chromatographic retention ofinsulin, the HIC, rPA and QFF steps should provide significant clearanceof insulin based on orthogonal modes of interaction. In addition,insulin, with a molecular weight of 5.8 kDa, should be cleared by thethree 30 kDa concentration/diafiltration steps in the harvest anddownstream process. The rPA step provides >3.0 logio clearance ofmethotrexate (MTX). In addition, MTX, with a molecular weight of 0.455kDa, should be cleared by the three 30 kDa concentration/diafiltrationsteps in the harvest and downstream process. Insulin and MTX are addedto the fermentation media in fixed amounts and are consumed as afunction of cellular metabolism during the 5000-L fermentation processprior to downstream processing. Insulin and MTX are measured at multiplepoints in the downstream process. The insulin and MTX levels at each ofthese points have been below the level of quantitation for past runscompleted.

Buffers: The objective of buffer preparation is to produce downstreamprocessing buffers that meet exemplary values, action limits and alertlimits. Consistent buffer quality is essential to ensure reproduciblechromatographic performance. The downstream steps of CTLA4-Ig ProcessCD-CHO1 require 17 buffers and solutions. Buffers and solutions areprepared and used for specific processing steps within a lot. Buffersthat have contact with the CTLA4-Ig in-process material are consideredto be significant process buffers. The product-contact buffers includeequilibration, wash, elution and product pool adjustment buffers. Thebuffers and solutions used in cleaning and sanitization steps andfunctional testing of the ultraviolet (UV) detectors in thechromatography skids have broad specification ranges. Due to the broadspecification ranges for these buffers and solutions and the absence ofproduct contact, no PPs are designated for these buffers and solutions.The PPs defined for the ten product-contact buffers and correspondingexemplary values are summarized. The maximum hold time limit for thesebuffers is three days, and is derived from the buffer vessel holdstudies₉, and supported by the buffer stability studies. Theproduct-contact buffers also have designated PPs with correspondingalert limits. The PPs and corresponding alert limits for these buffersare presented. Buffers and solutions that do not come into contact withCTLA4-Ig in-process material have designated PPs with alert or actionlimits and are presented. Non product-contact buffers and solutions arenot tested for endotoxin because they are either sanitization solutionsor chromatography resin cleaning buffers or solutions that are followedby column sanitization steps.

Q Sepharose Extreme Load Anion Exchange Chromatography Step

Anion exchange chromatography is performed using QXL resin from GEHealthcare (formerly Amersham Biosciences). A 1.0 m inner diametercolumn is packed with QXL resin to a height of 17 to 24 cm, representinga volume of 133 to 188 L. The column is qualified for use by determiningthe height equivalent to a theoretical plate (HETP) and asymmetry(A_(s)) of the packed column. A HETP of 0.02 to 0.08 cm and an A_(s) of0.8 to 1.2 are required for qualification of the QXL column. The QXLcolumn functions to capture the CTLA4-Ig protein from the in-processharvest material. The QXL capture step also accomplishes a volumereduction for further downstream processing. The QXL column operation iscarried out at ambient temperature. The clarified cell culture broth isloaded onto an equilibrated QXL column. The QXL chromatography step isperformed using a maximum flow rate of 28 L/min. The column inletpressure is maintained below 35 psig. The maximum abatacept protein loadfor the QXL column is 28 grams of abatacept per liter of resin. Thecolumn is prepared by equilibration with 25 mM HEPES, 100 mM NaCl, pH8.0 buffer. Following column equilibration, the harvest material isloaded onto the column with continuous monitoring of the UV absorptionof the effluent at 280 nm. Following load application, the column iswashed with 25 mM HEPES, 120 mM NaCl, pH 8.0 buffer. The CTLA4-Igprotein is eluted from the column with 25 mM HEPES, 850 mM NaCl, pH 7.0buffer. The table directly below shows process parameters for the QSepharose Extreme Load Chromatography Step.

Setpoint/ Parameter Target Value Action Limits Acceptance CriteriaProtein Load^(a,b) N/A^(c) N/A ≤28 g/L_(resin) Product Pool N/A N/A <1cfu/mL Bioburden^(a) Product Pool N/A N/A ≤50 EU/mL Endotoxin^(b,d)

Harvest hold time ensures process consistency with alert limitsestablished. The pre-filtration product pool bioburden parameter isassigned interim alert and action limits of <10 cfu/mL and <100 cfu/mL,respectively. The six QXL PPs defined by action limits are columnheight, flow rate, wash buffer conductivity, elution peak end opticaldensity (OD), step yield and load bioburden. The column height range wasestablished to provide sufficient volume of resin to capture the productfrom the harvest material. The action limits for this parameter wereestablished from data. Flow rate is an important factor to ensure theconsistency and performance of a chromatographic step. It is defined forthe QXL step as a PP with a maximum action limit. The wash bufferconductivity is established to remove weakly bound impurities from theQXL resin. Scale-down ranging studies determined that this parameter isan important factor in maintaining the consistency of the QXL step. Theelution peak end OD is defined to minimize the level of CTLA4-Ig HMWmaterial in the product pool. CTLA4-Ig HMW material elutes at the end ofthe elution peak. The step yield action limits ensure processconsistency for the QXL step. The action limits for flow rate, elutionpeak end OD and step yield PPs were established. Load bioburden isassigned an action limit of <1 cfu/mL. Twelve of the QXL PP are definedby alert limits as presented.

Buffer pH and conductivity values in the process are monitored. Buffersnot meeting pH and conductivity exemplary values at the time ofpreparation are rejected and the buffer lot discarded. For the QXL step,equilibration buffer conductivity and pH, wash buffer pH, and elutionbuffer conductivity and pH are defined. Column inlet pressure ensuresconsistency during the bind-and-elute chromatography step. The QXL stepis perfomed at a maximum pressure limit of 35 psig. The pressure limitof 35 psig is employed to prevent compression of the QXL resin inaccordance with the manufacturer's specification. Product poolconductivity is a PP with an alert limit. Product pool conductivity isassigned an alert limit to ensure that the CTLA4-Ig HMW material andCHOP in the QXL product pool bind effectively to the subsequent HICcolumn. The alert limits of 58.5 to 69.1 mS/cm were established. Productpool titer, sialic acid (SA) N-acetylneuraminic acid (NANA) molar ratio,CTLA4-Ig HMW material and CHOP are assigned alert limits in order toensure process consistency. Product pool titer and SA alert limits wereestablished.

The in-process material from the harvest operation step is loaded ontothe QXL column. The column is washed with a minimum of 10 CV of washbuffer (25 mM HEPES, 120 mM NaCl, pH 8.0), and the absorbance at 280 nm(A₂₈₅) of the column effluent is measured at the end of the wash step.The abatacept is then eluted from the column with a 25 mM HEPES, 850 mMNaCl, pH 7.0 buffer. The eluate is diverted into a collection vesselwhen the A280 increases to ≥0.02 absorbance units (AU) above the AUvalue at the end of the wash step. The eluate is collected until theA280 of the trailing edge of the elution peak decreases to a value of≤1.0 AU. Elution buffer is added directly to the eluate collectionvessel to achieve a target weight of 600±10 kg of the eluate. Anagitation rate of 30±5 rpm in the eluate collection vessel is used toensure that the contents are well-mixed. After a mixing period of ≥5minutes, the contents of the collection vessel are filtered through a0.2 μm cellulose acetate filter directly into a holding vessel. Anadditional ε 50 kg of elution buffer is added to the collection vesseland then filtered through the 0.2 μm cellulose acetate filter into thesame holding vessel. The contents of the holding vessel are stored at 2°to 8° C. for up to 72 hours.

TABLE 5 Process Parameters for the Q Sepharose Extreme LoadChromatography Step Setpoint/ Parameter^(a) Target Value Alert LimitAction Limit Harvest Hold Time^(b) N/A^(c) ≤10 hours ≤24 hoursPrefiltration Product N/A <10 cfu/mL <100 cfu/mL Pool Bioburden^(d)Column Height^(e) N/A N/A 17-24 cm Flow Rate 15-28 L/min N/A ≤28 L/minWash Buffer N/A N/A 11.0-15.0 mS/cm Conductivity^(f) Elution Peak 1.00AU^(g) N/A 0.97-1.03 AU^(h) End OD Step Yield N/A N/A 69-107% LoadBioburden^(i) N/A N/A <1 cfu/mL Equilibration Buffer N/A 10.7-12.7 mS/cmN/A Conductivity Equilibration Buffer N/A 7.8-8.2 N/A pH Wash Buffer pHN/A 7.7-8.3 N/A Elution Buffer N/A 69.8-77.1 mS/cm N/A ConductivityElution Buffer pH N/A 6.7-7.3 N/A Load Time^(j) N/A ≤6 hour N/A ColumnInlet N/A ≤35 psig N/A Pressure Product Pool N/A 58.5-69.1 mS/cm N/AConductivity^(k) Product Pool Titer NA ≥2.0 g/L N/A

Phenyl Sepharose Fast Flow Hydrophobic Interaction Chromatography Step

The primary objective of the HIC step is to reduce the level of CTLA4-Ighigh molecular weight species (e.g., tetramer) present in the QXLproduct pool. The CTLA4-Ig tetramer does not bind to the HIC resin underthe loading conditions used for the HIC step.

The HIC step is performed using a Phenyl Sepharose Fast Flow resin fromGE Healthcare. The HIC column binds CTLA4-Ig HMW material and CHOP,thereby reducing their concentrations in the CTLA4-Ig protein stream.The HIC column is prepared by equilibration with 25 mM HEPES, 850 mMNaCl, pH 7.0 buffer. The CTLA4-Ig product pool from the QXL step isapplied to the equilibrated column. Following load application, 25 mMHEPES, 850 mM NaCl, pH 7.0 buffer is applied to the column. CTLA4-Ig iscollected from the column in the flowthrough and column chase fractions.The HIC column is operated in multiple cycles to process a single lot ofCTLA4-Ig depending on the total mass of CTLA4-Ig in the QXL productpool. The column is cleaned and sanitized between cycles and lots.

The HIC load bioburden PP is defined by alert and action limits. The HICload bioburden PP is assigned interim alert and action limits of <10cfu/mL and <100 cfu/mL, respectively. The five HIC values defined forthe column are column height, flow rate, chase end OD, step yield andload hold time. The column height was determined to provide sufficientresin volume to process the elution pool from the QXL column in one, twoor three cycles per lot. The action limits for this parameter wereestablished from data. Flow rate is an important factor to ensure theconsistency and performance of a chromatographic step. It is defined forthe HIC step as a process parameter with a maximum action limit. Thechase end OD is defined to minimize the level of CTLA4-Ig HMW materialin the product pool. CTLA4-Ig HMW material elutes at the end of theproduct peak. Process step yield ensures process consistency for the HICstep. The action limits for flow rate, chase end OD and step yield PPswere established. Action limits for load hold time ensure processconsistency. The action limit for the load hold time PP of ≤5 days issupported by the product vessel hold time studies and the biochemicalstability study. Nine HIC PPs are defined by alert limits as presented.Buffer pH and conductivity parameters in the downstream process areidentified as PPs with alert limits. Buffers not meeting pH andconductivity exemplary values at the time of preparation are rejectedand the buffer lot discarded. For the HIC step, the conductivity and pHof the equilibration/chase buffer are defined as PPs with alert limits.

Column inlet pressure ensures consistency during the chromatographystep. The HIC step is performed at a maximum pressure limit of 13 psig.The pressure limit of 13 psig is employed to prevent compression of theHIC resin in accordance with the manufacturer's specification. Theproduct pool titer and SA alert limits ensure process consistency andwere established in the PAR reporti2. Product pool DNA, CHOP and MCP-1are assigned alert limits at this step in order to facilitate thequantification of their removal in the subsequent rPA step.

TABLE 7 Process Parameters for the Phenyl Sepharose Fast FlowHydrophobic Interaction Chromatography Step Setpoint/ Parameter^(a)Target Value Alert Limit Action Limit Load Bioburden^(b) N/A^(c) <10cfu/mL <100 cfu/mL Column Height^(d) N/A N/A 18-22 cm Flow Rate 7.6-18L/min N/A ≤18 L/min Chase End OD 1.0 AU^(e) N/A 0.8-1.0 AU^(e) StepYield N/A N/A 55-79% Load Hold Time N/A N/A ≤5 days Equilibration ChaseN/A 71.5-75.5 mS/cm N/A Buffer Conductivity Equilibration/Chase N/A6.7-7.3 N/A Buffer pH Load Tank 22° C. 19-25° C. N/A Temperature ColumnInlet Pressure N/A ≤13 psig N/A Product Pool Titer N/A ≥1.0 g/L N/AProduct Pool SA N/A 6.8-11.4 N/A NANA Molar Ratio Product Pool CHOP^(f)N/A ≤6600 ng/mL N/A Product Pool DNA^(f) N/A ≤45,000,000 pg/mL N/AProduct Pool MCP-1^(g) N/A ≤5600 ng/mL N/A ^(a)Information was obtainedfrom Table 2 in PAR Final Report: Purification¹².

Viral Inactivation Step: Inactivation of potential adventitious viralagents in the product pool from the HIC step is achieved by the additionof 20% Triton X-100 to a final concentration of 0.5% (v/v). Thedetergent-treated solution is mixed and held at 22±3° C. for one to fourhours before proceeding to the next step. The five PPs defined for theVI step are presented. The upper limit of the 20% Triton X-100 additionparameter is 3.8%. See the Table directly below showing processparameters for the viral inactivation step.

Setpoint/Target Acceptance Parameter Value Action Limit Criteria 20%Triton X-100 2.5% N/A^(c) 1.3-3.8% Addition^(a,b) (volume % of (volume %of HIC HIC pool) pool) Duration of Mixing^(d) N/A N/A^(c) ≥20 minutesAgitation Rate^(d) 30 rpm 25-35 ≥20 rpm Product Pool N/A N/A <1 cfu/mLBioburden^(b) Product Pool N/A N/A ≤5.0 EU/mL Endotoxin^(b,e)

TABLE 9 Process Parameters for the Viral Inactivation Step Parameter^(a)Setpoint/Target Value Alert Limit Action Limit Tank Temperature 22° C.19-25° C. 2-25° C. at Triton X-100 Addition^(a,b) Load Hold Time^(c)N/A^(d) N/A ≤5 days Triton X-100 N/A N/A 1-4 hours Incubation Time^(a,b)Step Yield^(a) N/A N/A 94-108% ^(a)Information was obtained from Table 3in PAR Final Report: Purification¹².

Recombinant Protein A Sepharose Fast Flow Affinity Step

Affinity chromatography is performed using an immobilized rPA resin fromGE Healthcare. The rPA chromatography step further purifies the CTLA4-Igprotein by reducing the levels of CHOP, MCP-1 and potential adventitiousviral agents. The affinity chromatography column is equilibrated with 25mM Tris, 250 mM NaCl, pH 8.0 buffer. After equilibration of the column,the Triton X-100 treated material from the VI step is applied to theaffinity chromatography column. The column is first washed with 25 mMTris, 250 mM NaCl, 0.5% Triton X-100, pH 8.0 buffer, followed by asecond wash with 25 mM Tris, 250 mM NaCl, pH 8.0 buffer. The CTLA4-Igprotein is eluted from the column with 100 mM Glycine, pH 3.5 buffer.The pH of the product pool from the affinity column is adjusted to7.5±0.2 with 2 M HEPES, pH 8.0 buffer. The four PPs defined for the rPAstep are presented in Table 10. The rPA chromatography step wasidentified as a viral clearance step, thus, column bed height wasestablished as a new PP with exemplary values of 21 to 25 cm. Productpool CHOP and product pool MCP-1 were previously identified as PPs forthe rPA step. These impurities were redefined as PPs with action limits.See the Table directly below showing process parameters for therecombinant Protein A Sepharose Fast Flow Chromatography Step.

Setpoint/Target Acceptance Parameter^(a) Value Action Limit CriteriaColumn Height^(b) N/A^(c) N/A 21-25 cm Protein Load N/A N/A^(c) ≤25g/L_(resin) Product Pool N/A N/A <1 cfu/mL Bioburden Product Pool N/AN/A ≤0.50 EU/mL Endotoxin^(d) ^(a)Information was obtained from Table 4in PAR Final Report: Purification¹².

Three PPs for the rPA step are defined by both alert and action limits,as presented. Load hold time ensures process consistency with alertlimits established in the PAR report. Action limits for load hold timeensure process consistency. The action limit for the load hold time PPof ≤48 hours is supported by the product vessel hold time studies andthe biochemical stability study. Ranging studies identified elutionbuffer pH as a significant factor affecting the level of CTLA4-Ig HMWmaterial in the rPA product pool. The alert limits for the elutionbuffer pH were established. The action limit for the elution buffer pHwas established from the scale-down ranging study. The rPA loadbioburden PP was assigned interim alert and action limits of <10 cfu/mLand <100 cfu/mL, respectively.

The seven rPA PPs defined by action limits are column inlet pressure,flow rate, step yield, product pool initial pH, product pool HMW,product pool CHOP and product pool MCP-1. The pressure limit of ≤13 psigis employed to prevent compression of the rPA resin in accordance withthe manufacturer's specification. Flow rate is an important factor toensure the consistency and performance of a chromatographic step. It isdefined for the rPA step as a PP with a maximum action limit. The actionlimits for step yield and product pool initial pH ensure processconsistency for the rPA step. Action limits for flow rate and productpool initial pH were established. The potential formation of CTLA4-IgHMW material during peak elution necessitates the definition of anaction limit of ≤2.5% for this parameter. The action limit range of 66to 108% was established using the mean±3 standard deviations data from asingle lot of rPA resin. This range is consistent with the step yieldsobserved in the resin lifetime study. The process-related impuritiesCHOP and MCP-1 were previously identified as PPs for the rPA step. Inthis report, these impurities were redefined as PPs with action limits.Product pool CHOP and MCP-1 are defined as CQAs with exemplary values toensure control of these process-related impurities. Twelve of the rPAPPs are defined by alert limits as presented. Buffer pH and conductivityparameters in the downstream process are identified as PPs with alertlimits. Buffers not meeting pH and conductivity exemplary values at thetime of preparation are rejected and the buffer lot discarded. For therPA step, equilibration/wash 2 buffer conductivity and pH, wash 1 bufferconductivity and pH, and elution buffer conductivity are defined as PPswith alert limits.

TABLE 11 Process Parameters for the Recombinant Protein A Fast FlowChromatography Step Setpoint/ Parameter^(a) Target Value Alert LimitAction Limit Load Hold Time^(b) N/A^(c) ≤43 hours ≤48 hours ElutionBuffer pH N/A 3.4-3.7 3.2-3.8 Load Bioburden^(d) N/A <10 cfu/mL <100cfu/mL Column Inlet Pressure N/A N/A ≤13 psig Flow Rate 6.7-9.6 L/minN/A ≤9.6 L/min Step Yield N/A N/A 66-108% Product Pool Initial N/A N/A≥5.8 pH Product Pool HMW N/A N/A ≤2.5% Product Pool CHOP N/A N/A ≤380ng/nL Product Pool MCP-1 N/A N/A ≤38 ng/mL Equilibration/Wash 2 N/A23.0-27.0 mS/cm N/A Buffer Conductivity Equilibration/Wash 2 N/A 7.8-8.2N/A Buffer pH Wash 1 Buffer N/A 22.2-27.4 mS/cm N/A Conductivity Wash 1Buffer pH N/A 7.7-8.2 N/A Elution Buffer N/A 0.5-1.5 mS/cm N/AConductivity Product Pool Final pH 7.5 7.3-7.7 N/A Product Pool TiterN/A ≥6.0 g/L N/A Product Pool SA N/A  8.0-11.0 N/A NANA Molar RatioProduct Pool Volume N/A 127-294 kg N/A after pH Adjustment^(e) ProductPool DNA N/A ≤47000 pg/mL N/A Product Pool Residual N/A ≤160 ng/mL N/ARecombinant Protein A^(f) Product Pool Triton X- N/A ≤4.0 μg/mL N/A100^(g) ^(a)Information was obtained from Table 4 in PAR Final Report:Purification¹².

Concentration/Diafiltration and Viral Filtration Step. Upon elution fromthe rPA column, the product pool is concentrated to achieve a targetvolume within a limit of CTLA4-Ig concentration. The concentrate is thensubjected to diafiltration with 25 mM HEPES, 100 mM NaCl, pH 8.0 bufferusing a UF system with 30 kDa nominal molecular weight cutoff (NMWC)membranes. Following diafiltration, a filter train is used to removepotential adventitious viral particles. The filter train consists of a0.2 μm filter, a 0.1 μm filter and 15 nm membrane filters (Planova 15Nfilter). Filters used in the VF step (0.2 μm, 0.1 82 m, and 15 nm) aresingle-use filters. The upper limit of the range for post-UF CTLA4-Igconcentration and Planova differential pressure were increased based onthe demonstrated viral clearance using these conditions. See table belowfor showing Process Parameters for the Viral Filtration Step.

Setpoint/Target Acceptance Parameter Value Action Limit Criteria Post-UFAbatacept N/A^(c) N/A 6.0-22 g/L Concentration^(a,b) Load Volume to ≤36kg/m² N/A ≤100 kg/m² Surface Area Ratio^(a) Planova Differential N/A N/A≤14 psid Pressure^(a,d) Product Pool N/A N/A <1 cfu/mL Bioburden^(e)Product Pool N/A N/A ≤0.50 EU/mL Endotoxin^(e,f)

TABLE 13 Process Parameters for the Viral Filtration Step Setpoint/Parameter^(a) Target Value Alert Limit Action Limit UF I Product PoolN/A^(c) <10 cfu/mL <100 cfu/mL Bioburden^(b) Difiltration Volumes N/AN/A ≥5.0 Step Yield N/A N/A 86-114% Load Hold Time^(d) N/A N/A ≤5 daysUF^(e) Feed Pressure N/A ≤35 psig N/A UF^(e) Retentate Flow N/A 2.5-7.5L/min N/A Filtrate Conductivity N/A 10.7-12.7 mS/cm N/A ^(a)Informationwas obtained from Table 5 in PAR Final Report: Purification¹².^(b)Information was obtained from BD-2005-706.0¹⁵.

Q Sepharose Fast Flow Anion Exchange Chromatography Step

The objective of the QFF chromatography step is to reduce the residualProtein A levels and provide additional reduction of host cell DNA fromthe viral filtration step product pool. The QFF column step is also usedto control the sialic acid to CTLA4-Ig molecules or protein molar ratioof the QFF chromatography step product pool and to provide additionalcontrol of the HMW material levels. The QFF anion exchangechromatography step also can remove residual recombinant protein A, hostcell DNA, Triton X-100 and potential adventitious viral agents.

Anion exchange chromatography is performed using QFF resin from GEHealthcare. The QFF column is equilibrated with 25 mM HEPES, 100 mMNaCl, pH 8.0 buffer. After column equilibration, the Planova filtrate isapplied to the QFF column. The column is first washed with 25 mM HEPES,120 mM NaCl, pH 8.0 buffer followed by a second wash with 25 mM HEPES,130 mM NaCl, pH 8.0 buffer. The CTLA4-Ig protein is eluted from thecolumn with 25 mM HEPES, 200 mM NaCl, pH 8.0 buffer.

Setpoint/Target Acceptance Parameter^(a) Value Action Limit CriteriaColumn Height^(a,b) N/A^(c) N/A 28-35 cm Protein Load^(a) N/A N/A ≤25g/L_(resin) Product Pool N/A N/A <1 cfu/mL Bioburden^(d) Product PoolN/A N/A ≤0.50 EU/mL Endotoxin^(a,d)

The QFF load bioburden value is <10 cfu/mL or <100 cfu/mL, respectively.The twelve QFF PPs defined by action limits are flow rate, wash 2 bufferconductivity, elution buffer conductivity, load hold time, step yield,and the levels of residual Protein A, DNA, Triton X-100, SA, HMW, CHOPand MCP-1 in the QFF product pool. Flow rate is a factor to consider inensuring the consistency and performance of a chromatographic step. Itis defined for the QFF step as a process parameter with a maximum actionlimit established in the PAR report. The conductivity values of the wash2 and elution buffers are PPs with action limits, because they aresignificant in determining step yield and product quality. The actionlimits for these parameters were determined during scale-down QFFranging studies. Action limits for load hold time ensure processconsistency. The action limit for the load hold time PP of ≤5 days issupported by product vessel hold time studies and a biochemicalstability study. Step yield ensures process consistency for the QFFstep. The quality parameters, product pool SA, HMW, CHOP and MCP-1 mustbe tightly controlled on the final chromatographic step. The actionlimits for the step yield, and the level of SA, CHOP and MCP-1 in theQFF product pool were established. The process-related impuritiesresidual protein A, DNA and Triton X-100 were previously identified asPPs for the QFF step. These impurities were redefined as PPs with actionlimits. Residual protein A, DNA and Triton X-100 are defined as CQAswith exemplary values to ensure control of these process-relatedimpurities. In this example, the action limit for product pool HMW wasrevised from ≤2.5 to ≤2.0%.

Buffer pH and conductivity parameters in the downstream process areidentified as PPs with alert limits. Buffers not meeting pH andconductivity exemplary values at the time of preparation are rejectedand the buffer lot discarded. For the QFF step, equilibration bufferconductivity and pH, wash 1 buffer conductivity and pH, wash 2 buffer pHand elution buffer pH are defined as PPs with alert limits. These limitswere established. Column inlet pressure ensures consistency during thebind-and-elute chromatography step. The QFF step is performed at amaximum pressure limit of 35 psig according to the manufacturer'sspecification. The parameters wash 1 buffer volume, wash 2 buffervolume, product pool titer and product pool volume are assigned alertlimits12 to ensure process consistency.

TABLE 15 Process Parameters for the Q Sepharose Fast Flow ChromatographyStep Setpoint/ Target Parameter^(a) Value Alert Limit Action Limit LoadBioburden^(b) N/A^(c) <10 cfu/mL <100 cfu/mL Flow Rate^(d) 4.8-8.7 N/A≤8.7 L/min L/min Wash 2 Buffer N/A N/A 12.8-15.2 mS/cm Conductivity^(e)Elution Buffer N/A N/A 18.5-20.9 mS/cm Conductivity^(e) Load HoldTime^(f) N/A N/A ≤5 days Step Yield N/A N/A 65-104% Product PoolResidual N/A N/A ≤9.5 ng/mL Recombinant Protein A Product Pool DNA^(g)N/A N/A ≤20 pg/mL Product Pool Triton N/A N/A ≤4.0 μg/mL X-100^(h)Product Pool SA N/A N/A 8.0-11.9   NANA Molar Ratio Product Pol HMW N/AN/A ≤2.0% Product Pool CHOP N/A N/A ≤95 ng/mL Product Pool MCP-1 N/A N/A≤9.5 ng/mL Equilibration Buffer N/A 10.5-12.9 mS/cm N/A ConductivityEquilibration Buffer NA 7.7-8.3 N/A pH Wash 1 Buffer N/A 12.4-14.4 mS/cmN/A Conductivity Wash1 Buffer pH N/A 7.7-8.3 N/A Wash 2 Buffer pH N/A7.7-8.3 N/A Elution Buffer pH N/A 7.7-8.3 N/A Column Inlet Pressure N/A≤35 psig N/A Wash 1 Buffer N/A 5.0-5.3 CV N/A Volume Wash 2 Buffer N/A5.0-5.4 CV N/A Volume Product Tool Titer N/A ≥0.65 g/L N/A Product PoolVolume N/A ≤800 kg N/A ^(a)Information was obtained from Table 6 in PARFinal Report: Purification¹².

Concentration/Diafiltration and Fill Steps. The product pool from theQFF anion exchange chromatography step is concentrated and subjected todiafiltration with 25 mM sodium phosphate, 50 mM NaCl, pH 7.5 bufferusing a UF system with 30 kDa NMWC membranes. The diafilteredconcentrate is filtered through a 0.2 μm filter into sterile containersand stored at 2 to 8° C. The drug substance may be frozen at −70° C. andstored at −40° C., if required. The single PP defined for theconcentration/diafiltration and fill step is presented. See the Tabledirectly below showing Process Parameters for the Drug SubstanceConcentration/Diafiltration and Fill Stens.

Action Parameters Setpoint/Target Limits Acceptance Criteria FinalConcentration^(a,b) 50 g/L 48-52 g/L 45-55 g/L

The UF II product pool bioburden PP is assigned interim alert and actionlimits of <10 cfu/mL and <50 cfu/mL, respectively. The six PPs definedby action limits for the concentration/diafiltration and fill step arediafiltration volumes, filtrate conductivity and pH, step yield, loadhold time, and process yield as presented. The lower limit ofdiafiltration volumes was established to ensure complete buffer exchangeof the CTLA4-Ig protein into the final drug substance buffer prior tothe fill step. Filtrate conductivity and filtrate pH further ensureconsistent drug substance formulation. Step yield ensures processconsistency for the concentration/diafiltration and fill step. Theaction limits for diafiltration volumes, filtrate conductivity and pH,and step yield were established. Action limits for load hold time ensureprocess consistency. The UF II load hold time of ≤5 days is supported bythe product vessel hold time studies and the biochemical stabilitystudy. The process yield action limit of 20 to 62% was recommended. TwoPPs are defined by alert limits to ensure process consistency for thestep, as presented.

TABLE 17 Process Parameters for the Drug SubstanceConcentration/Diafiltration and Fill Steps Parameters^(a) Setpoint AlertLimit Action Limit UF II Product Pool N/A^(c) <10 cfu/mL <50 cfu/mLBioburden^(b) Diafiltration Volumes N/A N/A ≥5.0 Filtrate ConductivityN/A N/A 8.3-10.3 mS/cm Filtrate pH N/A N/A 7.3-7.7   Step Yield N/A N/A 73-110% Load Hold Time^(d) N/A N/A ≤5 days Process Yield^(e) N/A N/A20-62% UF Feed Pressure N/A ≤35 psig N/A UF Retentate Flow N/A 2.5-7.5L/min N/A ^(a)Information was obtained from Table 7 in PAR Final Report:Purification¹².

Example 30 Drug Substance Final Fill Step

After completion of the concentration and diafiltration II step ofCTLA4-Ig, the CTLA4-Ig drug substance is filled from the 300-Lbioprocess bag into pre-sterilized 2-L and 10-L Biotainer PC bottleswithin a Class 100 environment.

The exterior of each bottle is disinfected with a 70% isopropanolsolution. The seal from around the cap of each bottle is removed and thebottle is tared prior to filling. A calculation is recorded in the batchrecord to ensure that the fill weight is between 500 grams to 1950 gramsfor 2-L bottles and between 7500 grams to 10200 grams for 10-L bottles.The drug substance is dispensed using a peristaltic pump into theBiotainer bottles through a single-use 0.45/0.2 μm filter and fillingbell. The bottles are capped and the caps tightened to a specifiedtorque setting. The cap of each filled bottle is sealed with tape andthe tape is initialed and dated. Each bottle is labeled with identifyinginformation as well as the date of fill, the sequential number of thefilled bottle within the lot and the initials of the operator.

During the filling process, air and surface microbial monitoring andparticle counts are performed. Drug substance samples are obtainedduring the filling operation. One sample is obtained prior to the fillof the first bottle for endotoxin testing. Additional samples forendotoxin testing are obtained in the middle and at the end of thefilling process. During the filling process, a sample is obtained fordrug substance release testing.

Example 31 Immunogenicity Studies

CTLA4-Ig is a soluble fusion protein that consists of the extracellulardomain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)linked to the modified Fc (hinge, CH2 and CH3 domains) portion of humanimmunoglobulin (Ig) G1. It is the first in a new class of agentsapproved for the treatment of rheumatoid arthritis (RA) that selectivelymodulates the CD80/CD86:CD28 co-stimulatory signal employed for fullT-cell activation. In RA, it is postulated that an unknown antigen ispresented via the major histocompatibility complex and activatesautoreactive T cells in the presence of a co-stimulatory signal.Subsequently, activated T cells recruit and activate downstream immunecells, orchestrating and perpetuating the cellular processes that leadto inflammation and joint destruction (Choy and Panayi (2001) N Engl JMed, 344(12):907-916).

Therapeutic recombinant biologic agents, such as CTLA4-Ig, can beimmunogenic and therefore have the potential to elicit an antibodyresponse. Immunogenicity against biological agents can theoreticallyimpact safety, efficacy and pharmacokinetics. Antibody-mediatedclearance of a biologic therapy may result in a reduction in druglevels, resulting in decreased efficacy. The antibody response may alsoprevent the drug from binding to its pharmacologic target, which willalso decrease efficacy. This can lead to a need for dose escalation overtime—so-called ‘dose creep.’ Dose creep has been reported with theprolonged use of the anti-TNF antibody, infliximab, in the treatment ofR A (Anderson (2005) Semin Arthritis Rheum, 34(5 Supp11):19-22) and thishas recently been noted as being a result of anti-infliximab antibodiesleading to a reduction in clinical efficacy in some patients (Haraoui etal., (2006) J Rheumatol, 33(1):31-36).

Here, the formation of anti-CTLA4-Ig and anti-CTLA-4 antibodies, andtheir potential effect on the efficacy and safety of CTLA4-Ig treatmentwere examined. Since CTLA4-Ig is likely to inhibit an immune response toitself, based on its activity as a selective co-stimulation modulatorand responses observed in non-clinical models, the effect of missing 1-2doses or discontinuing therapy on the level of immunogenicity was alsoexamined. Finally, seropositive samples were tested for neutralizingantibody activity.

Materials and Methods

Studies Evaluated

To determine whether CTLA4-Ig induces an immunogenic response inpatients with RA, an antibody response was assessed across multiplePhase II and III clinical trials, comprising six DB and four OL studyperiods in 2,237 RA patients, which included patients who had inadequateresponses to methotrexate (MTX) or anti-TNF therapy. One study (PhaseIIa) also assessed CTLA4-Ig for the treatment of RA as monotherapy(Table 40). Samples were generally evaluated pre-study, throughouttreatment, and 56 and/or 85 days following the last dose to allow timefor clearance of CTLA4-Ig.

TABLE 40 Overview of the studies included in this evaluation. No. ofpatients randomized and treated with CTLA4-Ig Phase Background Patientsin 10 mg/kg Other Study Study anti-rheumatic comparator or fixed dosesNo. design therapy group dose (mg/kg) Total Trials in RA patients withan inadequate response to MTX Phase II Randomized, dose- Days 1-180: 119115 105 (2.0)  339 IM101 100 ranging, placebo- MTX (10-30 mg/wk)controlled, DB trial Day 181-360: in patients with Adjustment active RAwhile on allowed (+1 MTX other non- biologic RA therapy) Phase IIIRandomized, Days 1-169: 219 433 0 652 IM101 102 placebo-controlled, MTX(10-30 mg/wk) DB in patients with Days 170-365: active RA while onAdjustment MTX allowed (+1 other non- biologic RA therapy) Trials in RApatients with an inadequate response to TNF blocking agents Phase IIIRandomized, Days 1-169: 133 258 0 391 IM101 029 placebo-controlled, Anynon- DB trial in patients biologic RA with active RA on therapybackground or anakinra DMARDs who have failed therapy with TNF-blockingagents due to lack of efficacy Safety study in RA Phase III Randomized,Days 1-85: 482 959 0 1441 IM101 031 placebo-controlled, Stable doses: DBsafety study in ±Non-biologic patients with RA RA therapy (with orwithout pre- ±Biologic RA existing therapy comorbidities) on Days86-365: background Adjustment DMARDs and/or allowed: biologics±Non-biologic RA therapy ±Biologic RA therapy Other supportive studiesPhase II Randomized, Days 1-180: 36  0 85 (2.0) 121 IM101 101placebo-controlled, ETAN (25 mg DB trial in patients 2x/wk) with activeRA Days 181-360: while on ETAN Adjustment allowed (±ETAN, +1non-biologic DMARD) Phase IIa Randomized, dose- None 32  33 26 (0.5) 122IM103 002 ranging, placebo- 32 (2.0) controlled, DB trial in RA patientswho failed at least 1 DMARD or ETAN; 85 days; follow-up through Day 169PK trials in healthy patients in RA program Phase II OL, uncontrolled,None 0  30 0 30 IM101 017 single-dose, PK study in healthy patients OLextensions in RA Phase II OL, uncontrolled Day 361+: 0  219* 0 219 IM101100 trial; 84, 68, and 67 MTX patients from ±1 Non- previous DB 10mg/kg, biologic RA 2 mg/kg, and therapy placebo arms, respectively PhaseII OL, uncontrolled Day 361+: ± 0  80* 0 80 IM101 101 trial; 58 and 22ETAN patients from ±1 Non- previous DB 2 mg/kg biologic RA and placebotherapy arms, respectively Phase III OL, uncontrolled Days 170+: 0  317*0 317 IM101 029 trial; 218 and 99 Any non- patients from biologic RAprevious DB 10 mg/kg, therapy and placebo or anakinra arms, respectivelyPhase III OL, uncontrolled Day 360+ 0  539* 0 539 IM1011 02 trial; 378and 161 MTX (10-30 mg/wk) OL patients from +1 non- previous DB 10 mg/kgbiologic RA and placebo therapy arms, respectively RA = rheumatoidarthritis; MTX = methotrexate; DB = double-blind; TNF = tumor necrosisfactor; DMARD = disease-modifying antirheumatic drug; ETAN = etanercept;OL = open-label; PK = pharmacokinetic *Subjects in the OL, uncontrolledperiods are a subset of those who completed the DB, placebo-controlledstudy periods

The incidence and type of prespecified peri-infusional adverse events(AEs), overall AEs and serious AEs (SAES), and discontinuations wereexamined in patients who developed a positive antibody response againstCTLA4-Ig or CTLA-4. The effect of immunogenicity on efficacy was alsoexamined by evaluating American College of Rheumatology (ACR) 20 andHealth Assessment Questionnaire responses in patients with a positiveantibody response.

Immunogenicity Assays

Basic assay formats: Because of high, pre-existing cross-reactivitydirected against the Fc portion of CTLA4-Ig in human serum, particularlyin RA populations, two direct-format enzyme-linked immunosorbent assays(ELISAs) were used to evaluate the antibody response. The anti-CTLA4-Igassay measured the antibody response to all portions of the molecule,but had lower sensitivity. The anti-CTLA-4 assay measured the antibodyresponse to the CTLA-4 portion only, removing the Ig region and thusconferring greater sensitivity. Both assays were used in either anendpoint titer (EPT) format (Assay A) or a screening format (Assay B).

Assay A Format: Phase II Double-Blind Clinical Immunogenicity AssayMethods

The anti-CTLA4-Ig assay and the anti-CTLA-4 assay used during Phase IIRA trials were collectively referred to as Assay A. In Assay A, CTLA4-Igor CTLA-4 was adsorbed onto 96-well microtiter plates that were thenincubated with test serum (3-fold serial dilutions starting at 1:10).Bound antibodies were detected using an alkaline-phosphatase conjugatedanti-human antibody cocktail (Southern Biotech, Birmingham, US) andvisualised using a p-NitroPhenyl Phosphate (PNPP) substrate. Since nohuman anti-CTLA4-Ig antibodies or positive control serum were available,these assays were validated using CTLA4-Ig-specific anti-sera generatedin a cynomolgus monkey. Results from each assay were expressed as EPTvalues. A patient was considered to have seroconverted when his/her EPTincreased by two or more serial dilutions (≥9-fold) relative to thatindividual's pre-dose (Day 1) EPT.

Assay B format: Phase III and Phase II Open-Label ClinicalImmunogenicity Assay Methods

For the Phase III trials, and 2-year Phase II OL periods, both theanti-CTLA4-Ig and anti-CTLA-4 assays were modified to reducenon-specific background, improve sensitivity, and the method todetermine positivity was changed and were collectively referred to asAssay B. These assays were also validated using CTLA4-Ig-specificantibodies purified from cynomolgus monkey anti-serum. For each ELISA,96-well microtiter plates coated with CTLA4-Ig (0.25 μg/mL) or CTLA-4(0.5 μg/mL), were incubated with test serum diluted 1:400 for 2 hours at22±5° C. (anti-CTLA4-Ig) or diluted 1:25 for 2 hours at 32-40° C.(anti-CTLA-4). After the primary incubation, bound antibodies weredetected with a horseradish-peroxidase (HRP) conjugated anti-humanantibody cocktail, followed by tetramethylbenzidine substrate.

Results for the anti-CTLA4-Ig assay were expressed as a post-/pre-ratiocalculated by dividing post-dose sample OD values by the correspondingpre-dose sample OD value analyzed on the same plate. Positivity wasbased on cut-off values established using placebo-treated RA patientsamples. If the ratio value was less than the cut-off, the sample wasconsidered negative and reported as a titer value <400. Any value thatexceeded this cut-off was considered conditionally positive.

Results for the anti-CTLA-4 assay were expressed as a ‘Ratio 1’ valuecalculated by dividing the mean patient serum sample OD by the mean ODof the negative control on the same plate. Positivity was based onvalues established using pooled serum from placebo-treated RA patientsas the negative control. If the value was less than the specifiedcut-off, the sample was considered negative and reported as a titervalue of <25. Any value that exceeded this cut-off was consideredconditionally positive.

Confirmatory Analyses

Conditionally positive samples identified in each assay (anti-CTLA4-Igand anti-CTLA-4) and in each assay format (Assays A and B), wereevaluated in an immunodepletion assay to determine specificity of theresponse. Anti-CTLA4-Ig positive samples were pre-incubated withapproximately 40 μg/mL of either CTLA4-Ig (the CTLA-4 portion of themolecule), another unrelated Ig fusion protein (CD40Ig) or an unrelatedprotein (ovalbumin) to identify the region of the molecule against whichthe anti-CTLA4-Ig reactivity might be directed (CTLA-4, Ig or junctionregion). Anti-CTLA-4 positive samples were similarly pre-incubated witheither CTLA4-Ig, the CTLA-4 portion or ovalbumin to confirm thespecificity of the anti-CTLA-4 reactivity. Following pre-incubation, allsamples were re-analyzed in the same original assay format describedabove. Samples where the pre-incubation resulted in ≥30% reduction in ODof the pre-incubated sample compared with the untreated sample, wereconsidered confirmed positives. If confirmed, samples were titrated toidentify the serum dilution that results in a ratio value equal to thecut-off of the particular assay and this value was reported as the EPT.

Neutralizing-Antibody Activity Assessments

A bioassay was conducted to assess the ability of patient samples withdrug-specific antibodies against CTLA-4 to inhibit or neutralize theactivity of CTLA4-Ig (inhibit binding to CD80/86), by preventing it frombinding CD80/86 on the T cell surface. Stable Jurkat T-celltransfectants expressing a luciferase gene under the control of theinterleukin (IL)-2 promotor were co-stimulated with Daudi B-cells in thepresence of anti-CD3 antibody. This co-stimulation, mediated through theinteraction between CD28 on the Jurkat T cell and CD80/86 on the Daudi Bcell in combination with anti-CD3 antibody, activates the IL-2 promoter,leading to increased transcription of the luciferase gene and, hence,increased luciferase protein expression. The luminescent signal ismeasured using a Luciferase Assay System. Since CTLA4-Ig blocks theCD80/86:CD28 interaction, adding CTLA4-Ig to the cell mixture blocksthis IL-2 promoter activation and decreases luminescence, whereaspre-incubation with a neutralizing antibody would restore theco-stimulation interactions and result in an increase in luminescence.

CTLA4-Ig neutralizing antibody activity was evaluated by determining theCTLA4-Ig response at concentrations of 0.1, 0.25 or 0.5 μg/mL in thebioassay in the presence of 1:25 post-dose seropositive serum andstatistically comparing it to the response in the presence of itscorresponding Day 1 sample. An anti-human CTLA-4 murine monoclonalantibody (11D4) with CTLA4-Ig-neutralizing activity in the bioassay wasused as a positive control in each analytical run. Owing to limitationsinherent in the bioassay test method, only post-dose samples withexisting levels of CTLA4-Ig ≤1 μg/mL could be evaluated, since higherdrug levels interfered with the neutralizing response, and furthersample dilution would decrease assay sensitivity.

Pharmacokinetic Evaluation

Population pharmacokinetic (POPPK) analysis was performed on serumsample data from patients from the DB periods of the Phase II/III trialswhere a positive immune response was confirmed. The validated POPPKmodel was applied to individual patient serum concentration data andmaximum a posteriori Bayesian estimates of individual PK parametervalues were obtained. The distribution of clearance, volume estimates,steady-state area under curve (AUC) values, and minimum concentration ofthe drug in the body after dosing (C_(min)) values for these patientswere compared with the distribution of these values in a larger data setof patients from the same trials who did not develop an immune response.

Results

Incidence of Anti-CTLA4-Ig and Anti-CTLA-4 Responses

A total of 2,237 patients had both pre-and post-baseline serum samplesand were eligible for assessment. Of these, 62 (2.8%) patients hadevidence of an anti-CTLA4-Ig or anti-CTLA-4 response, as determinedusing Assay A or B (FIG. 38). No patients demonstrated an immuneresponse to both the Fc and CTLA-4 domains of CTLA4-Ig. Three patientshad a response to the junction region. When the more sensitive Assay Bwas used, an antibody response to CTLA4-Ig was detected in 60 of 1,990patients (3.0%) (FIG. 38).

Of the patients evaluated in the Phase III studies (n=1,764), 203discontinued CTLA4-Ig therapy during the DB or OL periods, or did notenter into the subsequent OL study period and had sera collected 56and/or 85 days after discontinuation of therapy. Of the 203 patients, 15(7.4%) had an immunopositive response to either CTLA4-Ig (wholemolecule; n=5, 2.5%) or CTLA-4 (n=10, 4.9%; Table 42). Of the remaining1,561 RA patients who completed the Phase III DB period and continuedinto OL treatment, 40 (2.6%) had a positive antibody response during theDB or OL periods: 33 (2.1%) to CTLA4-Ig and 7 (0.4%) to CTLA-4.Interestingly, in the Phase IIa study of CTLA4-Ig as monotherapy, nopatients seroconverted for CTLA4-Ig or the CTLA-4 portion of themolecule; however, the less sensitive Assay A format was employed.

A total of 191 patients had a more than 30-day period without CTLA4-Igbetween their participation in the DB and OL periods. Of these, 3 (1.6%)patients had a positive antibody response to CTLA4-Ig and 1 (0.5%)patient had a positive antibody response to CTLA-4 during the OL period(Table 41). Sera were also analyzed from 587 RA patients who missed 1-2doses of study medication and restarted at any point during the study.Of these patients, 15 (2.6%) demonstrated a positive antibody responseto CTLA4-Ig and seven (1.2%) had a positive antibody response to CTLA-4(Table 41).

TABLE 41 Number (%) of seropositive patients with interrupted use ofCTLA4-Ig Number positive Description of interruption responses/numberevaluated (%) in scheduled CTLA4-Ig use Anti-CTLA4-Ig Anti-CTLA-4 TotalMissed 1-2 doses and 15/587  7/587 22/587 restarted use of CTLA4-Ig(2.6) (1.2) (3.7) >30 days without CTLA4-Ig 3/191 1/191  4/191 betweenDB and OL periods (1.6%) (0.5%) (2.1%) Discontinued during Phase III5/203 10/203  15/203 of the DB (sera collected 56 (2.5) (4.9) (7.4) and85 days after dosing) CTLA-4 = cytotoxic T-lymphocyte-associatedantigen-4; DB = double-blind; OL = open-label

Effect of Concomitant Methotrexate on Immunogenicity

A total of 2451 patients received concomitant MTX and 493 patients didnot. Overall, the percentage of patients with a positive antibodyresponse to CTLA4-Ig was generally similar whether they were receivingconcomitant MTX or not (2.3% vs 1.4%) (Table 42).

TABLE 42 Number of patients (%) with anti-CTLA4-Ig or anti-CTLA-4responses with or without receiving concomitant methotrexate. Number ofpositive Concomitant patients/number evaluated (%) TreatmentAnti-CTLA4-Ig Anti-CTLA-4 Total Methotrexate 40/2451 16/2451 56/2451(1.6) (0.6) (2.3) No 2/493 5/493 7/493 Methotrexate (0.4) (1.0) (1.4)CTLA-4 = cytotoxic T-lymphocyte-associated antigen-4

Impact of Immunogenicity on the Safety and Efficacy of CTLA4-Ig

The rates of AEs, SAES, peri-infusional AEs for all positive patientswere assessed and no relationship between immunogenicity and safety wasobserved. Similarly, no relationship between immunogenicity and efficacywas noted; however, interpretation of these data is restricted due tothe limited number of patients who seroconverted.

Neutralizing Activity of Anti-CTLA-4 Antibodies

Twenty-four serum samples from 20 patients were confirmed positive foranti-CTLA-4 reactivity in the anti-CTLA-4 antibody screening assay. Ofthese, 14 samples (collected from 13 patients) met the exemplary values(≤1 μg/mL CTLA4-Ig) for evaluation in the neutralization bioassay. Ofthese 13 samples, 1 was positive at Day 56 and 10 were positive at Day85 post-dose. Nine of the 14 samples (taken from 8 patients) exhibitedneutralizing antibody activity. With the exception of septicemia in onepatient, there were no medically significant AEs reported in thesepatients at, or near, the time of seroconversion that were consideredrelated or possibly related to therapy in these eight patients. Efficacydata were not collected during the period following studydiscontinuation (56 and 85 days after discontinuation), a period whenthe predominant number of samples were suitable for evaluation ofneutralizing antibodies. As such, it was not possible to evaluate theeffects of neutralizing antibodies on efficacy.

Pharmacokinetic Evaluation

Pharmacokinetic parameters were estimated for 31 of the 32 patients whohad a positive antibody response during the DB period of the PhaseII/III trials. Sera samples for PK analysis were not necessarilycollected on the day that a positive immune response was documented.Population PK modeling of patient data from the DB study periodssuggested that the predicted PK parameters in the 31 immunopositivepatients were comparable to those in a larger population of patients(n=386) without a positive immune response. Trough serum concentrationson the study day during the DB period when seroconversion was documentedranged from 1.16-24.21 μg/mL, with the majority of serum concentrationsbetween 5-20 μg/mL. Seroconversion did not appear to affect serum troughlevels. Distribution of clearance and volume of central compartment byimmunogenicity status is shown in FIG. 39.

MSD Electrochemiluminescence Assay. In an effort to improve thesensitivity of the binding immunogenicity assay and the ability todetect antibodies in the presence of drug (drug tolerance), a newgeneration of immunogenicity assays is being developed to monitoranti-drug antibodies to CTLA4-Ig by employing the Meso-Scale Discovery(MSD) technology. This new technology uses a label that emits light uponelectrochemical stimulation initiated at the electrode surface of amicroplate. The MSD format has been shown to have improved sensitivityand a better ability to detect antibodies in the presence of drugcompared to the ELISA format. This solution-phase technology allows thelabeled drug to more efficiently compete with the drug in the serum, andhas a greater dynamic range, signal to noise ratio, and increasedsurface capacity over the ELISA format. Unlike the current ELISAs, whichuse either the CTLA4 portion of the molecule or the whole molecule asthe capture reagent, the new assay is a bridging assay that employ abiotinylated and ruthenium-labeled CTLA4-Ig molecule that is incubatedwith patient samples prior to being added to an avadin-coated MSD plate.The electrochemiluminescence signal emitted by the ruthenium tag ismeasured using an MSD instrument. Positive samples, based on thevalidated assay cut-point, will be further evaluated by immunodepletionwith either CTLA4-Ig, CTLA4-T, or CD40Ig in the MSD assay to confirmpositivity and demonstrate to what portion of the CTLA4-Ig molecule theimmunogenicic response is directed, and endpoint titer is defined.

Example 32 Pharmacokinetic Parameters in Monkeys

Six female cynomolgus monkeys per group were administered a singleintravenous 10-mg/kg dose of CTLA4-Ig produced from the CD-CHO1 process.A control group of six female monkeys received saline (1 ml/kg). Toassess bioactivity of CTLA4-Ig, all monkeys were immunizedintramuscularly with 10 mg/animal of the T-cell-dependent antigenkeyhole limpet hemocyanin (KLH) within 30 min prior to dosing.

Animals were observed for 6 weeks following treatment. Blood sampleswere obtained predose; at 3 and 30 min; at 1, 2, 4, 8, 24, and 48 hr;and on days 4, 8, 11, 15, 22, 29, 36, and 43 postdose to determine andcompare the pharmacokinetic profiles. In the pharmacokinetic report,these study days correspond to days 0, 1, 2, 3, 7, 10, 14, 21, 28, 35,and 42, respectively. Serum samples were analyzed for CTLA4-Ig by aELISA method. A comparable blood sample was collected from controlanimals on the same days and, when appropriate, used to assess theanti-KLH antibody response. Assessment of the formation of CTLA4-Ig-specific antibodies was performed on serum obtained fromCTLA4-Ig-treated animals prestudy and weekly thereafter. KLH-specificantibody formation was determined on serum samples obtained from allanimals prior to immunization and approximately weekly thereafter for 4weeks postimmunization. Additional exemplary values for evaluationincluded survival, clinical signs, physical examinations (includingneurologic, respiratory rate and auscultation assessments), bodyweights, body temperatures, and food consumption.

Clinical-pathology evaluations were conducted prestudy and on day 45.All animals were returned to stock following completion of the study.

TABLE 43 Pharmacokinetic parameters of CTLA4-Ig produced from a processof the invention. BMS-188667 CLT Process Cmax AUC(0-T)^(b) T-HALF (mL/h/Vss (Lot Number) (μg/mL) (μg · h/mL) (H) kg) (mL/kg) CD-CH01 330.2219916.75 121.47 0.50 73.40 (Lot #MQJ611)  (53.52)  (3123.04)  (19.57)(0.08) (12.59)

Drug-specific antibody responses occurred on or after day 29 in three ofsix monkeys treated with the CD-CHO1-process material (Table 43; FIG.40). Minimal increases (20%) in blood urea nitrogen (BUN) and decreases(14%) in serum potassium in monkeys treated with the CD-CHO1 processmaterial were not physiologically or toxicologically meaningful becausevalues were only marginally outside historical control ranges and, forBUN, a concomitant increase in serum creatinine was not present. Noother changes in clinical pathology parameters were noted. Markedsuppression of the KLH antibody response (≥94% of the peak controlresponse) was observed in monkeys administered the CD-CHO1 processmaterial. Immunogenicity was markedly delayed until CTLA4-Ig serumlevels fell below immunosuppressive levels of approximately 1 μg/mL onor after day 29.

Clinical Pathology. Blood samples were collected from the femoral veinof fasted animals prior to dosing (day -13) and following the lastpharmacokinetic bleeding (day 45). Urinalyses were performed on urinecollected over an 18-hr period prestudy (day −13) and following the lastpharmacokinetic sampling (day 45). The following analytical parameterswere determined: Hematology: Hemoglobin, hematocrit, erythrocyte count,mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscularhemoglobin concentration, reticulocyte count, total and differentialleukocyte counts, platelet count, and evaluation of cell morphology inperipheral blood smears were determined. Coagulation: Prothrombin time,activated partial thromboplastin time, and plasma fibrinogen weredetermined. Serum Chemistry: Urea nitrogen, creatinine, glucose, totalcholesterol, total protein, albumin, globulins, albumin/globulin ratio,alanine aminotransferase, aspartate aminotransferase, alkalinephosphatase, total bilirubin, triglycerides, gamma glutamyltransferase,sodium, potassium, calcium, chloride, and phosphorus were determined.Urinalysis: Output, specific gravity, pH, color and appearance, andqualitative determinations of glucose, protein, ketones, bilirubin,occult blood, and urobilinogen were determined. Urinary sediments wereexamined microscopically.

Specific Antibody Response. Drug-specific antibody responses ofcomparable magnitude occurred in three of six monkeys treated with theCD-CHO1 process material, on or after day 29. As expected,immunogenicity with the process was markedly delayed until CTLA4-Igserum levels fell below immunosuppressive levels of approximately 1μg/mL on or after day 29. The mean CTLA4-Ig-specific antibody responsesfor monkeys given CD-CHO1 process material became positive on day 36 andpeaked on day 43 (the last time point assessed). Peak antibody titers inindividual monkeys given the CD-CHO1 process materials ranged from 23 to10,448.

Example 33 On-Column Disaggregation

The disaggregation process across an affinity chromatography resin isuseful and has applicability to all IgG-Fc based recombinant moleculesproduced in mammalians cells. Chaotropes can be used to disrupt highmolecular weight protein or material. This disaggregation can be doneon-column for any such protein. This example provides a method forperforming the disaggregation process on an exemplary material: i.e.,CTLA4-Ig.

CTLA4-Ig high molecular weight (HMW) material produced in the productionbioreactor can be partially converted into the functional CTLA4-Ig dimerby the use of chaotropic agents either in solution (batch mode) or inconjunction with the affinity chromatographic step (on-column mode).This process is referred to as “disaggregation.” Based on analyticalcharacterization studies of disaggregated CTLA4-Ig material, thedisaggregated material appears to be biochemically comparable to controlmaterial not subjected to disaggregation process.

In a fermentation process producing CTLA4-Ig molecules of the presentinvention, approximately 20 to 25% of the resulting CTLA4-Ig protein canbe in the form of HMW material (i.e., aggregate). Recovering a portionof this material as a functional dimer, therefore, has the potential ofan overall process yield enhancement of >10%.

Batch Mode Disaggregation Process

Disaggregation has been demonstrated in batch mode (in solution) bytreatment with moderate concentrations of chaotropic agents such asguanidine hydrochloride or urea followed by rapid dilution into a lowsalt refolding buffer to help the molecule gain its native conformation.

CTLA4-Ig purified using Protein A (resin used MAbSelect) was adjusted toa final concentration of ˜4-5 mg/ml to be used as a starting materialfor these batch experiments. This was then contacted in a 1:1 volumeratio with 2× concentrated chaotropic buffers to achieve a finalchaotrope concentration of 1-3 M (for Guanidine Hydrochloride) and 2-7 M(for Urea). Guanidine Hydrochloride concentrations >2 M and Ureaconcentrations >=4 M were effective in causing disaggregation ofCTLA4-Ig HMW material. The mobile phase used for these experiments was aphosphate buffered system at a pH range of 6.5-7.0. The disaggregationreaction was quenched by rapid dilution (in a volume ratio of 1:5) intoa refolding buffer consisting of 50 mM Tris, 25 mM NaCl, pH 8.5. Inbatch disaggregation experiments, 50 to 60% of the HMW material isconverted to CTLA4-Ig dimer with a >95% step yield. The decrease in thelevel of HMW material observed following the disaggregation step isshown in FIG. 41.

On-Column Disaggregation Process

To overcome potential tank and mixing limitations during scale-up of thebatch disaggregation process, a process combining the Protein A capturestep and the disaggregation step was evaluated. This process involvedusing the chaotropic solution as the elution buffer for the Protein Astep followed by collection of the elution pool into therefolding/dilution buffer.

Similar performance in disaggregation efficiency was observed using thebatch and on-column processes. The on-column disaggregation processwould have the distinct advantage of decreasing the number of processingsteps. Experimental details for the Protein A step using the on-columndisaggregation step are summarized in the following table. Resin used:MAbSelect (from GE Healthcare); Column bed height: 20 -25 cm

Residence time Step Buffer Buffer volume (min) Equili- 25 mM >3 CV 5bration phosphate, To be continued until the effluent 150 mM NaCl, pH &conductivity are close those pH 7.5 of the equilibration buffer or untilthere is no further change in pH and conductivity with each progressivecolumn volume of buffer used Column Harvested cell >30 g/l of resin 5load culture fluid Wash 25 mM >3 CV 5 phosphate, To be continued untilabsorbance 150 mM NaCl, has returned to <0.2 AU pH 7.5 Elution 25 mMPeak collection initiated when 10  phosphate, absorbance reaches 0.2 AUabove 2.65M GdHCl, baseline pH 6.5 (±0.2) Peak collection ended whenabsorbance returns to 0.2 AU above baseline Peak 50 mM Tris, The elutionpeak is to be N/A dilution 25 mM NaCl, immediately collected into the pH8.5 dilution buffer in a volume ratio of 1:5. Column 0.1N NaOH ~3 CV 5cleaning Column 20% Ethanol ~3CV 5 storage

Feasibility of Incorporation into the Downstream Process

A sample 3-column purification train was performed to generate finalprocess material with and without use of an on-column Protein Adisaggregation step. The chromatographic step yields obtained from thetwo purification trains are provided in Table 44.

TABLE 44 Chromatographic Yields for the 3 column process with andwithout Disaggregation Step Yield (%) 3-column 3-column process processwith On-column Process step (control) Disaggregation step Protein A 9595 HIC 60 69 AEX 84 87 Overall Chromatographic Yield 48 58

The incorporation of the disaggregation step in a sample 3-columnprocess resulted in an approximately 10% improvement in process yield.The product pools from the two process sequences were analyzed toevaluate the biochemical comparability of the resulting CTLA4-Igmaterial.

The N-glycan analyses by MALDI-TOF of the two product pools are shown inFIG. 42. Based on this analyses, the material appears comparable. ThisMALDI-TOF result was confirmed using an HPLC-based N-glycan assaymethod. The HPLC analyses demonstrated a <1.7% difference in thebiantenarry sialic acid peak between the control and disaggregated finalprocess material.

Tryptic peptide mapping was also performed on the two product pools toquantify the percent of deamidated and oxidized peptides present in thepurified material. The results (summarized in Table 45) demonstratedcomparable deamidation and oxidation levels for the two samples.

TABLE 45 Peptide Map Results T26 deamidation T6 oxidation site siteSample (% area) (% area) Control 3-column process 0.76 0.35 3-columnprocess with 0.69 0.33 Disaggregation step Standard material (5 g/L)0.94 0.37

Additionally, the B7-binding assay results were 101% and 98% for thecontrol and on-column disaggregated material, respectively.

A method for disaggregating IgG-Fc based recombinant molecules, such asthose produced by mammalians cells, comprising the step of contacting acomposition comprising such molecules in aggregated form with achaotropic agent (such as guanidine hydrochloride or urea) in an amountand for a time sufficient to disaggregate at least a portion of suchaggregated molecules, optionally followed by contacting saiddisaggregated portion of molecules with a refolding and/or quenchingagent (such as by rapid dilution into a low salt refolding buffer tohelp such molecules gain native conformation). Contact with thechaotropic agent can, for example, be carried out in batch, semi-batchor continuous mode, as well as, for example, in solution (such as inbatch mode), or in conjunction with a chromatographic step, such asduring an affinity chromatographic step (on-column mode).

A process combining an on-column purification, such as a Protein Acapture step, with the aforementioned disaggregation method can enhanceoverall process efficiency and is another embodiment of the invention.Therefore, the present invention contemplates a method fordisaggregating IgG-Fc based recombinant molecules, such as thoseproduced by mammalians cells, comprising the step of contacting acomposition comprising such molecules in aggregated form with achaotropic agent (such as guanidine hydrochloride or urea) in an amountand for a time sufficient to disaggregate at least a portion of saidaggregated molecules, wherein said contacting occurs on a chromatographycolumn, such as where said chaotropic agent is employed in solution forelution of said column (such as the elution buffer for a Protein Acolumn), optionally followed by contacting said disaggregated portion ofmolecules with a refolding and/or quenching agent (such as by rapiddilution into a low salt refolding buffer to help the molecule gainnative conformation).

The composition to be contacted with such chaotropic agent can compriseIgG-Fc based recombinant molecules in forms other than an aggregatedform (such as single chain forms or dimers), in addition to comprisingsaid molecules in aggregated form.

Exemplary IgG-Fc based molecules can include glycoproteins such as theCTLA4-Ig molecules of the present invention.

Example 34 Pharmacokinetics

A Phase 2B, Multi-Center, Randomized, Double-Blind, Placebo-ControlledStudy To Evaluate The Safety And Clinical Efficacy Of Two DifferentDoses of CTLA4-Ig Administered Intravenously To Subjects With ActiveRheumatoid Arthritis While Receiving Methotrexate): In this study,subjects received CTLA4-Ig at 2 different doses (2 and 10 mg/kg) orplacebo in combination with MTX. CTLA4-Ig was produced according to aprocess of the invention, and supplied in individual vials containing200 mg of CTLA4-Ig. CTLA4-Ig was administered IV to subjects on Days 1,15, and 30, and every 30 days thereafter for a year. Multiple dose PKwas derived from the serum concentration vs time data obtained duringthe dosing interval between Days 60 and 90 from subjects who wereenrolled into a site-specific PK substudy. For the subjects in the PKsubstudy, blood samples were collected before dosing on Day 60, and fora PK profile beginning on Day 60 at 30 minutes (corresponding to the endof CTLA4-Ig infusion), at 4 hours after the start of infusion, andweekly thereafter until Day 90. A total of 90 subjects were enrolled toparticipate in the PK substudy. However, complete PK profiles betweenthe dosing interval from Day 60 to 90 were obtained from 29 subjects (15subjects dosed at 2 mg/kg; 14 subjects dosed at 10 mg/kg).

A summary of the PK parameters is presented in Table 46. The resultsfrom the study showed that both Cmax and AUC(TAU), where TAU=30 days,increased in a dose proportional manner. For nominal doses increasing inthe ratio of 1: 5, the geometric means of Cmax increased in the ratio of1:5.2, while the geometric mean for AUC(TAU) increased in the ratio of1:5.0. In addition, T-HALF, CLT, and Vss values appeared to beindependent of dose. In these RA subjects, the mean T-HALF, CLT, and Vssvalues were around 13 days, ˜0.2 mL/h/kg, and ˜0.07 L/kg, respectively.The small Vss indicates that CTLA4-Ig is confined primarily to theextracellular fluid volume. Based on the dosing schema of dosing at 2and 4 weeks after the first infusion, then once a month thereafter,steady-state conditions for CTLA4-Ig were reached by the third monthlydose. Also, as a result of the dosing schema, serum concentrations wereabove steady-state trough concentrations during the first 2 months oftreatment. Comparison of the trough (Cmin) values at Days 60, 90, and180 indicated that CTLA4-Ig does not appear to accumulate followingmonthly dosing. The mean Cmin steady-state values for all subjectsreceiving monthly IV doses of 2 and 10 mg/kg CTLA4-Ig ranged between 4.4to 6.7 μg/mL and 22.0 to 28.7 μg/mL, respectively.

TABLE 46 Summary of Multiple PK studies in Rheumatoid Arthritis SubjectsPharmacokinetic Parameters of Abatacept Geometric Mean # Age: Treatment(% CV) Mean (SD) Study Study Subjects (Mean Dose Cmax AUC (TAU) T-HALFCLT Vss Objective Design (M/Fem) range) (mg/kg) (μg/mL) (μg · h/mL)(Days) (mL/h/kg) (L/kg) Assess the Randomized 29 54  2.0  54.9 (29) 9573.5 (30) 13.5 (5.9) 0.23 0.07 efficacy, double- (18/11) (34-83) (N =15) (0.13) (0.04) safety, blind, 10.0 284.2 (23) 47624.2 (31) 13.1 (5.3)0.22 0.07 multiple dose placebo- (N = 14) (0.09) (0.03) PK andcontrolled, immunogenic multiple potential of dose study. intravenously30-minute administered IV infusion doses of abatacept

In RA patients, after multiple intravenous infusions, thepharmacokinetics of CTLA4-Ig showed proportional increases of C_(max)and AUC over the dose range of 2 mg/kg to 10 mg/kg. At 10 mg/kg, serumconcentration appeared to reach a steady-state by day 60 with a mean(range) trough concentration of 24 (1-66) mg/mL. No systemicaccumulation of CTLA4-Ig occurred upon continued repeated treatment with10 mg/kg at monthly intervals in RA patients.

Population pharmacokinetic analyses in RA patients revealed that therewas a trend toward higher clearance of CTLA4-Ig with increasing bodyweight. Age and gender (when corrected for body weight) did not affectclearance. Concomitant methotrexate (MTX), nonsteroidalanti-inflammatory drugs (NSAIDs), corticosteroids, and TNF blockingagents did not influence CTLA4-Ig clearance.

Example 35 Determination of Molar Ratio of Mannose, Fucose, andGalactose by CE

A capillary electrophoresis method as been developed for thequantitative analysis of neutral monosaccharide content in LEA2CTLA4^(A29YL104E)-Ig. Neutral monosaccharides, including mannose,fucose, and galactose are released from CTLA4^(A29YL104E)-Ig samples byacidic hydrolysis at a high temperature condition (2M trifluoroaceticacid, 6 hours at 95° C.). The released neutral monosaccharides are thenfluorescently labeled with aminopyrene trisulfonic acid (APTS), in thepresence of acetic acid as a catalyst, and NaBH₃CN as a reducing reagent(67 mM APTS, 330 mM HAc, 83 mM NaBH₃CN, 3 hours at 55° C.). Xylose isadded to each sample and serves as an internal standard. Ratio of thepeak area of each neutral monosaccharide against that of the internalstandard is utilized for quantitation.

Reagents: Hydrolysis solution (2M trifluoroacetic acid (TFA));Derivatization solution I (0.1M 8-amino-1,3,6, trisulfonic acic,trisodium salt (APTS) aqueous solution); Derivatization solution II(0.25M NaBH₃CN in 1M acetic acid); Running buffer (60±5 mM sodiumtetraborate, pH9.25); Capillary rinsing solutions (1N NaOH; 1N HCl; 80%methanol); Monosaccharide standard stock solutions of mannose (Man),fucose (Fuc), galactose (Gal), and xylose (Xyl) at concentration of 10mg/ml; Monosaccharide working solution I: Internal standard workingsolution is 100 fold dilution of Xyl stock solution; Monosaccharideworking solution II: Neutral mix standard working solutions, 100 folddilution of Man, Fuc and Gal stock solutions.

Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector:Beckman laser induced (LIF) detection system coupled with P/ACE MDQ);Uncoated capillary (i.d. 25 μm, o.d. 360 μm) 27-31 cm total length toaccommodate P/ACE MDQ.

Capillary Electrophoresis running conditions: Running buffer (60 mMsodium tetraborate, pH 9.25); Capillary cartridge temperature: 25° C.;Voltage: 25-30 kV, positive mode; Detector condition: LIF detector,excitation at 488 nm, emission at 520m; Sample injection: pressureinjection mode, 20s at 0.5PSI; Run time: 10 min; Sample storage: 10° C.

Hydrolysis: 10 μL of Xylose working solution and 200 μL of 2M TFA weremixed to make the system blank. 10 μL of Xylose working solution and 10μL of Neutral mix standard solution were mixed with 200 μL of 2M TFA tomake the monosaccharide standard. 10 μL of Xylose working solution and10 μL of sample (for example, CTLA4^(A29YL104E)-Ig, approximately 1mg/ml) were mixed with 200 μL of 2M TFA to make the test sample. Alltubes were vortexed for 10 sec, and centrifuge for 10 sec, followed byincubation at 95° C. for 6 hours. After the hydrolysis step the sampleswere places at −20° C. for 10 min to cool down. Samples were spun downfor 10 sec and evaporated to dryness in SpeedVac.

Derivatization: Samples were reconstituted with 10 μL of Derivatizationsolution I. Sample was briefly mixed, and 5 μL of Derivatizationsolution II was added. Samples were loaded in a pre-warmed centrifugeand incubated for 3 hours at 55° C. while centrifuging at 2000 rpm.

CE injection: The final volume of the samples after derivatization wasbrought to 100 μL by addition of HPLC grade water, and 10 μL of sampleswere transferred to a CE micro vial with 190 μL HPLC grade water. Beforesample injections the CE cartridge was rinsed extensively with HPLCgrade water (1-3 min run time), followed by an equilibrating rinse withrunning buffer (5 min run time). Following the initial rinse,monosaccharide standards and samples for analysis were injected in theCE cartridge (15 min run time). Following the injection run of eachstandard or test sample, the CE cartridge was rinsed and equilibratedwith HPLC grade water and running buffer (Table 51). Theelectopherograpm of the system suitability should be similar to FIG. 44wherein peak 1 is mannose; peak 2 is xylose; peak 3 is fucose; and peak4 is galactose.

TABLE 51 Instrument Method Time Event Value Duration Summary DescriptionRinse - 40.0 psi 3.00 min forward Water rinse Pressure Rinse - 40.0 psi5.00 min forward Running buffer Pressure rinse Inject - 0.5 psi 20.00sec override, Injection Pressure forward  0.00 min Separate - 30 kV15.00 min 0.17 min ramp, Separation Voltage normal polarity  0.05 minAuto Zero 15.00 min Stop Data 15.00 min End

System Suitability

The electropherogram of the system suitability should be similar to thatshown in FIG. 44, where peak 1 is mannose; peak 2 is xylose; peak 3 isfucose; and peak 4 is galactose.

When CE instruments other than the Beckman MDQ system are used, thelength of the capillary may be different from that specified in thismethod. This will cause variations in analyte migration time, as well aspeak intensity. But the peak pattern of monosaccharide analytes shouldremain the same.

Resolution between two neighbor peaks for the first System Suitabilitystandard can be calculated according to the following equation:

R=2(t ₂ −t ₁)/(W ₁ +W ₂)

-   -   Where,    -   R: resolution    -   t₂, t₁: migration times of the two neighbor peaks respectively    -   W₁, W₂: peak widths at baseline of the two neighbor peaks        respectively

R value must be ≥1.0. If R <1.0, rinse the capillary using thewashing/rinse sequences. If the problem persists, replace old bufferwith freshly prepared run buffer or replace the capillary.

For the last System Suitability injection, the last peak (galactose)must have a tailing factor <1.4 using the following formula:

T=W_(0.05)/2f

-   -   Where: T: tailing factor    -   W_(0.05): width of peak at 5% of height    -   f: width of the peak front at peak maximum

If T ≥1.4, rinse the capillary with the washing/rinse sequences; if theproblem persists, replace old buffer with freshly prepared runningbuffer or replace the capillary. Peak Area Ratio of galactose and xylosemust have an RSD of ≤10%. The migration time of galactose needs to be≤15.0 minutes. The electropherogram profile should be equivalent to FIG.44.

The monosaccharide standard percent RSD can be determined by comparingpeak area ratios of internal standard and monosaccharide standardcomponents via dividing the peak area for each monosaccharide componentby the peak area of the internal standard for each monosaccharidestandard injection. The percent RSD can be calculated for mannose,fucose, and galactose. The RSD should be ≤10%.

Determination of the Molar Ratios of Neutral Monosaccharides to Protein

Peak area ratios of neutral monosccahrides (for example, Man, Gal andFuc) relative to internal standard Xylose can be calculated according tothe formulas provided below in order to determine the molar ratios ofeach neutral monosaccharide to protein. For example, the peak area ratiois equal to a monosaccharide peak area (Gal, Fuc or Man) divided by theXylose peak area, wherein the relative standard deviation (RSD) for thepeak area ratio is equal or less that 10%. The following equations canbe used to calculate the following:

For molar ratio of Mannose/Protein:

$R_{man} = \frac{A_{man} \times A_{{xyl}\; 0} \times V_{{man}\; 0} \times C_{{man}\; 0} \times M\; W_{LEA29Y}}{A_{xyl} \times A_{{man}\; 0} \times V_{p} \times C_{p} \times 180.2}$

Where,

-   -   R_(man): molar ratio of mannose vs. protein    -   A_(man): peak area (μV·sec) of mannose in sample    -   A_(xyl): peak area (μV·sec) of xylose in sample    -   A_(xyl0): peak area (μV·sec) average of xylose in monosaccharide        standard    -   A_(man0): peak area (μV·sec) average of mannose in        monosaccharide standard    -   V_(man0): volume of mannose contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(man0): concentration of mannose contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   V_(p): volume of protein sample used for hydrolysis (in μL)    -   C_(p): concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(LEA29Y): Molecular weight of LEA29Y (or        CTLA4^(A29LY104E)-Ig) (91,232 Da)    -   MW of mannose: 180.2 daltons.

For molar ratio of Fucose/Protein:

$R_{fuc} = \frac{A_{fuc} \times A_{{xyl}\; 0} \times V_{{fuc}\; 0} \times C_{{fuc}\; 0} \times M\; W_{{LEA}\; 29\; Y}}{A_{xyl} \times A_{{fuc}\; 0} \times V_{p} \times C_{p} \times 164.2}$

Where,

-   -   R_(fc): molar ratio of fucose vs. protein    -   A_(fuc): peak area (μV·sec) of fucose in sample    -   A_(xyl): peak area (μV·sec) of xylose in sample    -   A_(xyl0): peak area (μV·sec) average of xylose in monosaccharide        standard    -   A_(fuc0): peak area average (μV·sec) of fucose in monosaccharide        standard    -   V_(fuc0): volume of fucose contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(fuc0): concentration of fucose contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   V_(p): volume of protein sample used for hydrolysis (in μL)    -   C_(p): concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(LEA29Y): Molecular weight of LEA29Y (or        CTLA4^(A29YL104E)-Ig) (91,232 Da)    -   MW of fucose: 164.2 daltons.

For molar ratio of Galactose/Protein:

$R_{gal} = \frac{A_{gal} \times A_{{xyl}\; 0} \times V_{{{gal}\; 0}\;} \times C_{{gal}\; 0} \times M\; W_{{LEA}\; 29\; Y}}{A_{xyl} \times A_{{gal}\; 0} \times V_{p} \times C_{p} \times 180.2}$

Where,

-   -   R_(gal) molar ratio of galactose vs. protein    -   A_(gal): peak area (μV·sec) of galactose in sample    -   A_(xyl): peak area (μV·sec) of xylose in sample    -   A_(xyl0): peak area (μV·sec) average of xylose in monosaccharide        standard    -   A_(gal0): peak area (μV·sec) average of galactose in        monosaccharide standard    -   V_(gal0): volume of galactose contained in monosaccharide        working solution used for hydrolysis (in μL)    -   C_(gal0): concentration of galactose contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   V_(p): volume of protein sample used for hydrolysis (in μL)    -   C_(p): concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(LEA29Y): Molecular weight of LEA29Y (or        CTLA4^(A29YL104E)-Ig) (91,232 Da)    -   MW galactose: 180.2 daltons.

TABLE 52 Average Molar Ratio of Monosaccharide to CTLA4^(A29YL104E)-Igprotein. MONOSACCHARIDE RANGE Mannose 11-23 Fucose 4.2-7.5 Galactose9.2-18

Example 36 Determination of Molar Ratio of GalNAc and GlcNAc by CE

A capillary electrophoresis method has been developed for thequantitative analysis of amino monosaccharide content inCTLA4^(A29YL104E)-Ig, a glycoprotein with 6 N-linked glycosylation sitesand at least 1 O-linked glycosylation site. Amino monosaccharides,including N-acetyl galactosamine (GalNAc) and N-acetyl glucosamine(GlcNAc) are released from CTLA4^(A29YL104E)-Ig sample by acidichydrolysis at a high temperature condition (4N HCl, 6 hours at 95° C.).The released amino monosaccharides go through a re-acetylation step byincubating with acetic anhydride on ice for half an hour. They are thenfluorescently labeled with aminopyrene trisulfonic acid (APTS), in thepresence of acetic acid as a catalyst, and NaBH₃CN as a reducing reagent(67 mM APTS, 330 mM HAc, 83 mM NaBH₃CN, 3 hours at 55° C.). N-acetylmannosamine is added to each sample and serves as an internal standard.Ratio of the peak area of each amino monosaccharide against that of theinternal standard is utilized for quantitation.

Reagents: Hydrolysis solution (4N HCl); Derivatization solution I (0.1M8-amino-1,3,6, trisulfonic acic, trisodium salt (APTS) aqueoussolution); Derivatization solution II (0.25M NaBH₃CN in 1M acetic acid);Re-acetylation buffer (25 mM sodium bicarbonate, pH9.5); Running buffer(60±5 mM sodium tetraborate, pH9.25); Capillary rinsing solutions (1NNaOH; IN HCl; 80% methanol); Monosaccharide standard stock solutions ofGalNAc, GlcNAc, and ManNAc at concentration of 10 mg/ml; Monosaccharideworking solution I: Internal standard working solution is 100 folddilution of ManNAc stock solution; Monosaccharide working solution II:Amino mix standard working solutions, 100 fold dilution of GalNAc andGlcNAc stock solutions.

Instrumentation: CE system is Beckman P/ACE MDQ CE sytem; Detector:Beckman laser induced (LIF) detection system coupled with P/ACE MDQ).

Capillary Electrophoresis running conditions: Running buffer (60 mMsodium tetraborate, pH 9.25); Capillary cartridge temperature: 25° C.;Voltage: 25-30 kV, positive mode; Detector condition: LIF detector,excitation at 488 nm, emission at 520 m; Sample injection: pressureinjection mode, 20s at 0.5PSI; Run time: 10 min; Sample storage: 10° C.

Hydrolysis: 10 μL of ManNAc working solution and 200 μL of 4N HCl weremixed to make the system blank. 10 μL of ManNAc working solution and 10μL of Amino mix standard solution were mixed with 200 μL of 4N HCl tomake the monosaccharide standard. 10 μL of ManNAc working solution and10 μL of sample (for example, CTLA4^(A29YL104E)-Ig sample, etc.;approximately 1 mg/ml) were mixed with 200 μL of 4N HCl to make the testsample. All tubes were vortexed for 10 sec, and centrifuge for 10 sec,followed by incubation at 95° C. for 6 hours. After the hydrolysis step,the samples were placed at -20° C. for 10 min to cool down. Samples werespun down for 10 sec and evaporated to dryness in SpeedVac.

Re-acetylation: Hydrolyzed and dried samples were reconstituted with 204of re-acetylation buffer and 44 of acetic anhydride, followed by mixingand with incubation on ice (30 min). Samples were spun down for 10 secand evaporated to dryness in SpeedVac. Sample were each reconstitutedwith 100 μl of HPLC grade water and were evaporated to dryness with aSpeedVac.

Derivatization: Reconstituted samples (10 μL of Derivatization solutionI HPLC) were provided 5μL of Derivatization solution II. After mixing,samples were loaded in a pre-warmed centrifuge and incubated for 3 hoursat 55° C. while centrifuging at 2000 rpm.

CE injection: The final volume of the samples after derivatization wasbrought to 100 μL by addition of HPLC grade water, and 10 μL of sampleswere transferred to a CE micro vial with 190 μL HPLC grade water. Beforesample injections the CE cartridge was rinsed extensively with HPLCgrade water (1-3 min run time), followed by an equilibrating rinse withrunning buffer (5 min run time). Following the initial rinse,monosaccharide standards and samples for analysis were injected in theCE cartridge (10 min run time). Following the injection run of eachstandard or test sample, the CE cartridge was rinsed and equilibratedwith HPLC grade water and running buffer. The electopherograpm of thesystem suitability should be similar to FIG. 45, wherein peak 1 isGalNAc; peak 2 is ManNAc; and peak 3 is GlcNAc.

TABLE 53 Instrument Method Time Event Value Duration Summary DescriptionRinse - 40.0 psi 3.00 min forward Water rinse Pressure Rinse - 40.0 psi5.00 min forward Running buffer Pressure rinse Inject - 0.5 psi 20.00sec override, Injection Pressure forward  0.00 min Separate - 30 kV10.00 min 0.17 min ramp, Separation Voltage normal polarity  0.05 minAuto Zero 10.00 min Stop Data 10.00 min End

System Suitability:

The electropherogram of the system suitability should be similar to thatshown in FIG. 45, where peak 1 is GalNAc; peak 2 is ManNAc; and peak 3is GlcNAc

When CE instruments other than the Beckman MDQ system are used, thelength of the capillary may be different from that specified in thismethod. This will cause variations in analyte migration time, as well aspeak intensity. But the peak pattern of monosaccharide analytes shouldremain the same.

Resolution between two neighbor peaks for the first System Suitabilitystandard can be calculated according to the following equation:

R=2(t ₂ −t ₁)/(W ₁ +W ₂)

-   -   Where,    -   R: resolution    -   t₂, t₁: migration times of the two neighbor peaks respectively    -   W₁, W₂: peak widths at baseline of the two neighbor peaks        respectively

R value must be ≥1.0. If R <1.0, rinse the capillary using thewashing/rinse sequences. If the problem persists, replace old bufferwith freshly prepared run buffer or replace the capillary.

For the last System Suitability injection, the last peak (GlcNAc) musthave a tailing factor <1.4 using the following formula:

T=W_(0.05)/2f

-   -   Where: T: tailing factor    -   W_(0.05): width of peak at 5% of height    -   f: width of the peak front at peak maximum

If T ≥1.4, rinse the capillary with the washing/rinse sequences; if theproblem persists, replace old buffer with freshly prepared runningbuffer or replace the capillary. Peak Area Ratios of GlcNAc and ManNAcmust have an RSD of ≤10%. The migration time of GlcNAc must be ≤10.0minutes. The electropherogram profile should be equivalent to FIG. 45.

The monosaccharide standard percent RSD can be determined by comparingpeak area ratios of internal standard and monosaccharide standardcomponents via dividing the peak area for each monosaccharide componentby the peak area of the internal standard for each monosaccharidestandard injection. The percent RSD can be calculated for GalNAc andGlcNAc. The RSD should be 10%.

Determination of the Molar Ratios of Amino Monosaccharides to Protein

Peak area ratios of Amino monosccahrides (for example, GalNAc andGlcNAc) relative to internal standard ManNAc can be calculated accordingto the formulas provided below in order to determine the molar ratios ofeach amino monosaccharide to protein. For example, the peak area ratiois equal to a monosaccharide peak area (GalNAc or GlcNAc) divided by theManNAc peak area, wherein the relative standard deviation (RSD) for thepeak area ratio is equal or less that 10%. The following equations canbe used to calculate the following

For molar ratio of GalNAc/Protein:

$R_{GalNAc} = \frac{A_{GalNAc} \times A_{{ManNAc}\; 0} \times V_{{GalNAc}\; 0} \times C_{{GalNAc}\; 0} \times M\; W_{{LEA}\; 29\; Y}}{A_{ManNAc} \times A_{{GalNAc}\; 0} \times V_{p} \times C_{p} \times 221.2}$

Where,

-   -   R_(GalNAc): molar ratio of GalNAc vs. protein    -   A_(GalNAc): peak area (μV·sec) of GalNAc in sample    -   A_(ManNAc): peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0): peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(GalNAc0): peak area (μV·sec) average of GalNAc in        monosaccharide standard    -   V_(GalNAc0): volume of GalNAc contained in monosaccharide        working solution used for hydrolysis (in μL)    -   C_(GalNAc0): concentration of GalNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   V_(p): volume of protein sample used for hydrolysis (in μL)    -   C_(p): concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(LEA29Y): Molecular weight of LEA29Y (or        CTLA4^(A29YL104E)-Ig) (91,232 Da)    -   MW GalNAc: 221.2 Daltons.

For molar ratio of GlcNAc/Protein:

$R_{GlcNAc} = \frac{A_{GlcNAc} \times A_{{ManNAc}\; 0} \times V_{{GlcNAc}\; 0} \times C_{{GlcNAc}\; 0} \times M\; W_{{LEA29}\; Y}}{A_{ManNAc} \times A_{{GlcNAc}\; 0} \times V_{p} \times C_{p} \times 221.2}$

Where,

-   -   R_(GlcNAc): molar ratio of GlcNAc vs. protein    -   A_(GlcNAc): peak area (μV·sec) of GlcNAc in sample    -   A_(ManNAc): peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0): peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(GlcNAc0): peak area (μV·sec) average of GlcNAc in        monosaccharide standard    -   V_(GlcNAc0): volume of GlcNAc contained in monosaccharide        working solution used for hydrolysis (in μL)    -   C_(GlcNAc0): concentration of GlcNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   V_(p): volume of protein sample used for hydrolysis (in μL)    -   C_(p): concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(LEA29Y): Molecular weight of LEA29Y (or        CTLA4^(A29YL104E)-Ig) (91,232 Da)    -   MW GlcNAc: 221.2 Daltons

TABLE 54 Average Molar Ratio of moles Monosaccharide to molesCTLA4^(A29YL104E)-Ig protein. MONOSACCHARIDE RANGE GalNAc 2.0-3.2 GlcNAc18-32

Example 37 N-Linked Oligosaccharide Carbohydrate Profiling ofCTLA4^(A29YL104E)-Ig by High Performance Anion Exchange Chromatography

The carbohydrate structures present on glycoproteins can affect theirfunction and in vivo clearance. It is therefore important to monitor thestructural consistency of the carbohydrates of recombinantly producedbatches of glycoproteins. Here, N-linked (asparagine-linked)carbohydrates present on CTLA4^(A29YL104E)-Ig are monitored. In thismethod, oligosaccharides are cleaved by enzymatic digestion with PNGaseF (Peptide: N-Glycosidase F), separated by high performance anionexchange chromatography (HPAEC), and monitored by electrochemicaldetection (integrated amperometry). The chromatogram generated is theN-linked carbohydrate profile, wherein profiles of CTLA4^(A29YL104E)-Igsamples should be similar to such.

Reagents for Mobile Phases for Isolation of Oligosaccharides by ReversedPhase and graphite carbon HPLC: Eluent 1 (0.05% Trifluoroacetic Acid(TFA) in HPLC grade water); Eluent 2: (0.05% TFA in 60% Acetonitrile(ACN), 40% HPLC Water (60:40, ACN: H₂O); Eluent 3: 0.05% TFA in 40%Acetonitrile, 40% Isopropanol (IPA), 20% HPLC Water (40:40:20, ACN:IPA:H₂O)).

Reagents for Preparation of Mobile Phases for HPAEC OligosaccharideCarbohydrate Profiling: Eluent 1: 500 mM Sodium Acetate (NaOAc); Eluent2: 400 mM Sodium Hydroxide (NaOH); Milli-Q Water; 4M Sodium Hydroxide(approximately 4M NaOH); 50 mM Sodium Phosphate Buffer, 0.02% SodiumAzide, pH=7.5; PNGase F Enzyme Working Stock in 50 mM Sodium PhosphateBuffer, 0.02% Sodium Azide, pH=7.5; Stachyose Stock Solution (1.25mg/mL); Stachyose System Suitability Standard (12.5 μg/mL).

INSTRUMENTATION AND CONDITIONS—(Equivalent instrumentation may besubstituted.)

Instrumentation:

Alliance HPLC system equipped Waters Corporation with a calibratedtemperature- controlled autosampler (37° C.), a Rheodyne switching valveapparatus and UV detector Synergi 6-column selector Phenomenex, (CatalogNo. AV0-6080) Column 1: Luna 5μ, C18(2) Phenomenex, (Catalog No. 4.6 ×150 mm 00F-4252-E0) Column 2: HyperCarb 5μ Phenomenex, (Catalog No. 4.6mm × 100 mm CH0-3301) Dionex DX-500 HPLC System Dionex Corporationincludes: GP50 Gradient Pump AS50 autosampler (refrigerated) EluentDegas Module Liquid Chromatography Module ED40 detector DX-LAN PeakNetSoftware (version 5.1 or upgrade) on suitable computer system Column:CarboPac PA-1 Dionex Corporation, (Catalog 4 × 250 mm No. 35391) GuardColumn: CarboPac PA-1 Dionex Corporation, (Catalog 4 × 50 mm No. 43096Millennium Data Collection Version 3.2 system

Sample Preparation: To a 1.7 mL Eppendorf tube, 80 μL of 50 mMNaPhosphate buffer containing 0.02% NaAzide, pH 7 and 80 μL of sample(˜25 μg/μL for a total of 2 mg of CTLA4^(A29YL104E)-Ig, etc.) were addedfollowed by 16 μL of 10× Denaturing Buffer (5% SDS, 10%β-Mercaptoethanol). Samples were mixed thoroughly and subsequentlyboiled for 2 minutes to denature proteins. Vials were cooled to roomtemperature, then 16 μL of NP40 (10%) and 40 μL of the PNGase F workingstock were added and subsequently mixed. After samples were incubated at37° C. for 24±2 hours, they were transferred into an HPLC autosamplervial, ready for injection on the HPLC instrument.

Chromatography Conditions for Oligosaccharide Isolation:

Column Temperature Ambient (22-25° C.) Flow Rate Program Initial to 30.0min 1.0 mL/min 30.0 to 35.0 min, 1.0-2.0 mL/min 35.0 to 40.0 min,2.0-1.0 mL/min 40.0 to 50.0 min, 1.0 mL/min 50.0 to 60.0 min, 1.0-0.1mL/min Gradient Program Mobile Phases and Time Gradient Conditions (min)%1 %2 %3 1: 0.05% TFA Initial 100 0 0 2: 0.05% TFA 15.00 80 20 0 inACN/H₂O (60:40) 3: 0.05% TFA in 15.01 0 100 0 ACN/IPA/H₂O (40/40/20)25.00 0 100 0 30.00 0 0 100 35.00 0 0 100 40.00 100 0 0 50.00 100 0 060.01 100 0 0 Autosampler Temperature 37° C. Injection Volume 100 pL RunTime 50 minutes Data Collection Time 50 minutes Column Switching Eventsfor 6 column switching valve and Waters Alliance Time Rheodyne apparatus(min) Event Function Initial Switch 1 Off Initial Switch 2 On InitialSwitch 3 On Initial Switch 4 Off 11.0 Switch 4 On 30.0 Switch 4 Off 30.0Switch 1 On 30.0 Switch 2 Off 40.0 Switch 2 On 40.0 Switch 1 Off

Chromatography Conditions For Oligo Profile by Anion-ExchangeChromatography:

Column Temperature Ambient (22-25° C.) Flow Rate 1 mL/min Mobile Phasesand Gradient Conditions Gradient Program 1: 500 mM NaOAc *the secondvalue for some gradient 2: 400 mM NaOH steps below indicate the extentto which 3: Milli-Q Water the gradient may be modified in order toadjust the retention time of the system suitability standard Time (min)%1 %2 %3 Initial 0 50-35 50-65 0.0 0 50-35 50-65 1.0 0 50-35 50-65 2.0 450-40 46-56 60.0 45 50 5 61.0 0 50 50 80.0 0 50 50 ED40 DetectorSettings Mode Integrated Amperometry Applied Potentials Time Pot. Integ.0.00 +0.05 0.20 +0.05 Begin 0.40 +0.05 End 0.41 +0.75 0.60 +0.75 0.61−0.15 1.00 −0.15 Range 200 nC Analog Output Setup Output = offset Zeroposition = 5% full scale Volts full scale = 1.0 v Rise time = 1 secPolarity = + Autosampler Temperature 4° C. Injection Volume 30 μL RunTime 80 minutes Approximate Retention Time (RT; minutes) according to RTof System Suitability (SS) Standard Approximate Retention Time (min) SS:9.6 Peak 1A: 10.5 Peak 1B: 11.5 Peak 1C: 12.5 Peak 1D: 13.5 Peak 1E:15.0 Peak 2: 24.5 Peak 3: 37.5

The CTLA4^(A29YL104E)-Ig samples can have a carbohydrate profiledepicted in the chromatogram of FIG. 46. The retention times of eachdomain, as identified in FIG. 46, should be approximately:

Domain I (Peak 1A, 1B, 1C, 1D and 1E) 10-17 min Domain II: 21-29 minDomain III: 33-43 min Domain IV: 48-56 min

Retention times are system dependent and should shift similarly asstachyose.

Calculations

Theoretical Plates (N): The number of Theoretical Plates (N) can bedetermined based on the Stachyose peak using the formula below. This isdone through the Millennium data analysis system or may also be donemanually.

N=16(t/W)²

-   -   Where:        -   t: retention time measured from time of injection to peak            elution time at maximum height.        -   W: width of peak by extrapolation of sides to baseline.

N must be ≥6000. If the plate count is less than 6000, the column shouldbe replaced.

Tailing Factor (T): The column Tailing Factor (T) can be calculatedbased on the Stachyose peak using the formula below. This is donethrough the Millennium data analysis system or may also be donemanually.

T=(W_(0.05)/2f)

-   -   Where:        -   W_(0.05): width of peak at 5% of height (0.05 h).        -   f: the measurement (width) from front edge of peak at            W_(0.05) to middle of peak at maximum height.

T must be ≤1.2. If the tailing factor is greater than 1.2, the buffercomposition should be evaluated, the column should be replaced orcleaned.

% Domain Area:

-   -   Domain I: Sum of the peak areas at approximate retention times        10-15 minutes (Peaks 1A-1E; for example, FIG. 46)    -   Domain II: Sum of the peaks from 21-27 minutes    -   Domain III: Sum of the peaks from 34 -40 minutes    -   Domain IV: Peak area for peaks at 45-56 minutes

${{Domain}\mspace{14mu} {Area}\mspace{14mu} \%} = {\frac{{Individual}\mspace{14mu} {Domain}\mspace{14mu} {Area}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {all}\mspace{14mu} {Domain}\mspace{14mu} {Areas}} \times 100\%}$

Percent Main Peak Area: The % Peak Area for each of the five major peakscan be calculated for the following using the equation below: Peaks 1A,1B, 1C, 2 (two non-resolved species), 3 (two non-resolved species), and4.

${{Peak}\mspace{14mu} {Area}\mspace{14mu} {Percent}} = {\frac{{Peak}\mspace{14mu} {Area}\mspace{14mu} {for}\mspace{11mu} {Individual}\mspace{14mu} {Peak}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {all}\; {DomainAreas}} \times 100\%}$

Example 38 Capillary Electrophoretic Identification ofCTLA4^(A29YL104E)-Ig in Drug Substance and Drug Product

CTLA4^(A29YL104E)-Ig is a water-soluble glycoprotein withimmunosuppressant activity. A capillary electrophoresis method using anuncoated fused silica capillary was used for the identification ofCTLA4^(A29YL104E)-Ig. Samples (for example, CTLA4^(A29YL104E)-Ig, etc.)are heated for 5 minutes at 70° C. and then analyzed immediately by UVdetection set at 214 nm to confirm identification.

Additionally, a 1:1 mixture of CTLA4^(A29YL104E)-Ig and CTLA4-Igmaterial was mixed and injected to confirm that the two proteins couldbe separated and differentiated. This method can distinguish betweenCTLA4^(A29YL104E)-Ig from CTLA4-Ig by comparing the migration time ofCTLA4^(A29YL104E)-Ig material to sample.

EQUIPMENT (Equivalent equipment may be substituted):

Capillary Electrophoresis Instrument Beckman P/ACE MDQ Detector ModuleUV at 214 nm Capillary Polymicro Technologies Inc., 360 μm o.d./75 μmi.d., Uncoated, (Part No. 2000019, TSP075375) Capillary window makerMicroSolve Technology, (Catalog No. 07200-S)

REAGENTS and SOLUTIONS: Running Buffer (14.4 mM sodium borate; 17.3 mMsodium dodecyl sulfate; 2.0% Acetonitrile); Phosphate Dilution Buffer(22.3 mM sodium phosphate, dibasic; 4.2 mM sodium phosphate, monobasic;53.4 mM sodium chloride); Borate Dilution Buffer (83.5 mM NaBO₂, pH9.6); Reference or Sample Working Solutions (10±1 mg/mL sample inphosphate dilution buffer); Reference or Sample Injection Solutions(10.0 μL sample+50 μL of borate dilution buffer); Sample 1:1CTLA4-Ig-CTLA4^(A29YL104E)-Ig Mixture Solution (10.0 μL CTLA4-Igsample+10.0 μL CTLA4^(A29YL104E)-Ig sample+50 μL of borate dilutionbuffer); Injection Solution.

Run Conditions:

UV Detector Wavelength 214 nm Data Rate 4 Hz Peak Width (points) 16-25Injection 10 seconds (0.5 psi) Total Length * ~60 cm Capillary EffectiveLength** ~50 cm Capillary Cartridge Temperature 15° C. Voltage 19 kV-23kV Run Time 15 minutes * Total Length: from capillary inlet to outlet**Effective Length: from capillary inlet to the detection window

The CTLA4-Ig sample migration time of the main peak is about 11.0±0.4minutes. The CTLA4^(A29YL104E)-Ig sample migration time of the main peakis about 12.0±0.4 minutes. The main peak migration times between eachsample should be at least 0.6 minutes apart (FIG. 47).

Example 39 Hydrolysis and HPLC Analysis for the N-Acetylneuraminic Acidand N-Glycolyl Neuraminic Acid Content Determination onCTLA4^(A29YL104E)-Ig

The degree of sialylation in recombinant proteins can affect thepharmacokinetics and rate of clearance. CTLA4^(A29YL104E)-Ig is arecombinant protein possessing both N-and O-linked carbohydrate sites.The glycans occupying these sites possess variable degrees ofsialylation. Besides structural heterogeneity of its sialylation, thecontent of individual sialic acid could vary from lot to lot. An overallmeasure of the ratio of sialic acid to protein is therefore obtained.

N-Acetyl Neuraminic acid (NANA) and N-Glycolyl Neuraminic Acid (NGNA)content present CTLA4^(A29YL104E)-Ig was examined. The sialic acids areliberated from the protein by acid hydrolysis and then fluorescentlylabeled with DMB. The labeled sialic acids are separated on an RP-HPLCC-18 column and quantitated from a response factor of a concurrently runsialic acid standard. Data analysis and report values (as molar ratio ofNANA or NGNA to protein) are specified within this example method. Thisexample describes the method used to determine the amount of N-AcetylNeuraminic acid (NANA) and N-Glycolyl Neuraminic Acid (NGNA) present inCTLA4^(A29YL104E)-Ig. The sialic acids are liberated from the protein byacid hydrolysis. The released NANA and NGNA are then fluorescentlylabeled with 1, 2,-diamino-4, 5-methylnoxybenzene (DMB). The labeledsialic acids are separated on a RP-HPLC C-18 column and detected byfluorescent detection (Ex=373 nm, Em=448 nm). NANA and NGNA arequantitated based on the response factors of a concurrently run NANA andNGNA standards. The test results are reported as molar ratio (MR) ofNANA and NGNA to protein respectively.

Reagents and Solutions: 1.0 M H₂SO₄; 50 mM H₂SO₄; Fluorescence LabelingReagent (18 mM sodium hydrosulfite (Na₂S₂O₄), 7% 2-mercaptoethanol, 7 mM1,2-diamino-4,5-methylene-dioxybenzene dihydrochloride (DMB)); MobilePhase Running Buffer A (20% Methanol); Mobile Phase Running Buffer B(70% Methanol); N-Acetyl Neuraminic Acid Standard Solution (1 mg/mL);N-Glycolyl Neuraminic Acid Standard Solution (1 mg/mL); SystemSuitability Standards (50 μL of the NANA or NGNA solutions in 900 μL ofwater); Sample Solutions (for example, 2 mg/ml of CTLA4^(A29YL104E)-Ig,etc.); NANA working Stock Standard Solution (dilute stock to 50 μg/mL);NGNA working Stock Standard Solution (dilute stock to 50 μg/mL).

Hydrolysis: 20 μL of each NANA standard, NGNA standard,CTLA4^(A29YL104E)-Ig, and system suitability standard solution wasaliquotted into separate 1.5 mL centrifuge tubes and 200 μL of 50 mMsulfuric acid solution was added to each vial. The contents were gentlymixed and incubated at 80EC for 1 hour ∀ 5 minutes. When hydrolysis iscomplete, the sampled were quickly centrifuged.

Fluorescence labeling: Fluorescence labeling reagent (200 μL) was addedto each sample and mixed thoroughly. Samples were then incubated in thedark at 80EC for 45 ∀ 5 and subsequently cooled.

Instrumentation and Chromatographic Conditions (Equivalentinstrumentation may be substituted):

Ternary Pump System Hewlett Packard Model 1090 RP C-18 HPLC Column,Jones Chromatography, 4.6 × 50 mm, 3μ (Catalog No. 4M5313) FluorescenceDetector Hewlett Packard Model 1046A Autosampler Hewlett Packard Model1090 equipped with refrigeration to 4° C. Integration SystemVG/Multichrom HPLC Chemstation Hewlett Packard (DOS Series)

Chromatographic Parameters

Flow: 1.0 mL/min Mobile Phase A: 20% MeOH/water Mobile Phase B: 70%MeOH/water Time % A % B Linear Gradient: 0.01 98 2.0 1.0 98 2.0 4.0 901.0 4.01 98 2.0 6.00 98 2.0 Injection Volume: 10 μL Run Time: 6 minColumn Temperature: Room Temperature Retention Time (NANA): 3.1 ∀ 0.5min Retention Time (NGNA): 2.3 ∀ 0.5 min Max. Pressure: 300 barExcitation Wavelength: 373 nm Emission Wavelength: 448 nm PMT Gain: 8

HPLC System: Waters 2690/2695 separations module or equivalent.Fluorescence Detector: Waters 2475 Multi wavelength FluorescenceDetector or equivalent. Data Acquisition: Waters Millennium 32 orEmpower. Column: Luna 5μ, C18, 100 A, 150 × 4.6 mm, Phenomenex, (CatalogNo. 00F-4252-E0) Digital Heat block WVR, (Catalog No. 13259-056) orequivalent. Mini Centrifuge WVR, (Model No. C-1200) or equivalent.Mobile Phase A: 10% (v/v) MeOH/90% water Mobile Phase B: 70% (v/v)MeOH/30% water Flow Rate: 1 mL/min Injection Volume: 10 μL Run Time: 30min Column Temperature: Room Temperature Excitation Wavelength: 373 nmEmission Wavelength: 448 nm Gain: 1

Elution Gradient:

Time Flow % A % B Curve Initial 1.0 85.0 15.0 * 20.00 1.0 85.0 15.0 620.50 1.0 0.0 100.0 6 25.00 1.0 0.0 100.0 6 25.50 1.0 85.0 15.0 6 30.001.0 85.0 15.0 6 35.00 0.05 0.0 100.0 11 

Sialic acid standards preparation (˜5 mM). N-Acetyl NeuraminicAcid(NANA, MW=309.3) Standard (˜5 mM). Accurately weigh 154.5±1.0 mg ofN-Acetyl Neuraminic Acid into a 100 mL volumetric flask. Dissolve andQ.S. to the volume with DI water, mix well. Aliquot the solution into 2mL cryogenic vials.

${{Conc}.\mspace{11mu} ({mM})} = \frac{{Wt}\mspace{11mu} ({mg}) \times P}{309.3 \times 100\mspace{11mu} ({mL})}$

P=Purity of NANA-from Vendor COA (i.e., 99%=0.99).

N-Glycolyl Neuraminic Acid (NGNA, MW=325.7) Standard (˜0.25 mM).Accurately weigh 8.0±1.0 mg of N-Glycolyl Neuraminic Acid into a 100 mLvolumetric flask. Dissolve and Q.S. to the volume with DI water, mixwell. Aliquot the solution into a 2 mL cryogenic vial.

${{Conc}.\mspace{11mu} ({mM})} = \frac{{{Wt}{\mspace{11mu} \;}({mg})} \times P}{325.7 \times 100\mspace{11mu} ({mL})}$

P=Purity of NGNA-from Vendor COA (i.e., 99%=0.99). The aliquoted sialicacid standards can be stored at −20° C. for up to six months.

Sialic acid standard mixture preparation. Sialic acid standard mixturefor system suitability and quantitation (50 μM NANA, 1 μM NGNA). Add 1mL of 5 mM NANA, 400 μl of 0.25 mM NGNA into a 100 mL volumetric flask.Q.S. to the volume with DI water and mix well. Aliquot the sialic acidstandard mixture into 2 mL cryogenic vials. The aliquoted sialic acidstandard mixture can be stored at −20° C. for up to six months.

Sample and reference material preparation. Thaw frozen protein samplesat 2-8° C., mix well. Dilute both samples and reference material toapproximate 0.5 mg/mL CTLA4^(A29YL104E)-Ig (e.g. Protein Conc.=25.0mg/ml, add 50.0 μl of protein into 2450 μl of water). Centrifuge thediluted test samples and reference material for 5 minutes at 10,000 rpmin order to remove particulates.

Hydrolysis. Blank: To a 2.0 mL centrifuge tube, add 50 μL of DI waterand 200 μL of 50 mM sulfuric acid. This serves as blank. Sialic acidstandard for system suitability and quantitation. To a 2.0 mL centrifugetube, add 50 μL of sialic acid standard mixture and 200 μL of 50 mMsulfuric acid. Prepare in duplicate. Denote as Std1 and Std2. ReferenceMaterial: To a 2.0 mL centrifuge tube, add 50 μL of dilutedCTLA4^(A29YL104E)-Ig reference material (˜0.5 mg/mL), and 200 μL of 50mM sulfuric acid. Prepare in duplicate, denote as RM1 and RM2. TestSamples: To a 2.0 mL centrifuge tube, add 50 μL of dilutedCTLA4^(A29YL104E)-Ig drug substance (˜0.5 mg/mL), and 200 μL of 50 mMsulfuric acid. Prepare in duplicate. Denoted as S1-1, S1-2; S2-1, S2-2;S3-1, S3-2; etc. Vortex samples for approximately 5 seconds andcentrifuge for approximately 5-10 seconds. Place samples in a heatingblock and incubate at 80° C.±5° C. for 1 hour ∇±5 minutes. Allow thehydrolyzed samples to cool to room temperature. Centrifuge hydrolyzedsamples briefly to force condensate into the tube (˜10 seconds at highspeed).

Derivatization. Pre-heat the heating block to 80° C.±5° C. Add 200 μL offluorescence labeling reagent to each hydrolyzed sample. Vortexapproximately 5 seconds and centrifuge for ˜10 seconds. Place samples inan 80° C.±5° C. heating block for 40±5 minutes. Cover the heating blockwith aluminum foil, as the labeling solution is light sensitive. Letderivatized samples cool to room temperature. Vortex and centrifugesamples for approximately 10 seconds to force condensate into the tube.

Preparation for injection. Ensure that the column is equilibrated withmobile phase prior to analysis. Transfer sufficient sample (100-200 μL)from each centrifuge tube into an autosampler vial with limited insert.A typical autosampler loading for 10 sample injections is as follows:

Sample# Description # of injections 1 Blank 1 2 Std1 2 3 Std1 2 4 Std2 25 RM1 1 6 RM2 1 7 S1-1 1 8 S1-2 1 9 S2-1 1 10 S2-2 1 11 S3-1 1 12 S3-2 113 S4-1 1 14 S4-2 1 15 Std1 1 16 Std2 1

Where Std1 and Std 2 are the preparations of Sialic Acid StandardMixture Solution; RM1 and RM2 are the preparations for control samples;and S is a sample injection. The first four (Sample#2 & 3) of SialicAcid Standard (Std1) injections will be used for System Suitabilitypurposes. The four injections of Sample#3 (Std1) and Sample#4 (Std2)will be used for calculation. For additional sample injections, repeatautosampler loading 5 to 16.

SYSTEM SUITABILITY. The chromatogram profile of the system suitabilitysamples should be similar to chromatogram shown in FIG. 48. For thefirst injection of System Suitability Standard (Std1), The USPresolutions (R) for NGNA and NANA must be ≥1.5. Four injections of theSystem Suitability Standard (Std1) must meet the following exemplaryvalues: The RSD of the peak area for NANA must be ≤5%. The RSD of thepeak area for NGNA must be ≤10%. The migration time of NGNA peak must beeluted at 11.3±2.0 minutes. The migration time of NANA peak must beeluted at 16.0±2.5 minutes. The RSD of peak area of four standardinjections, (Std1 , Sample#3) and (Std2, Sample#4) must be ≤10%. The RSDof peak area of all bracket standard injections from the sequence mustbe ≤15%.

Preparation of HPLC System: Columns were equilibrated with 98% Buffer Aand 2% Buffer B at 1 mL/min for 15 minutes. 10 μL of fluorescentlylabeled system suitability solution was injected into the system. Peakresolution and the number of theoretical plates can then be calculatedusing the following equations:

$R = \frac{2\mspace{11mu} \left( {{T\; 2} - {T\; 1}} \right)}{{W\; 2} + {W\; 1}}$

-   -   Where: R=Resolution    -   T1=Retention time (min) of N-glycolyneuaminic acid    -   T2=Retention time (min) of N-acetylneuraminic acid    -   W1=Peak width at baseline of N-glycolylneuraminic acid (min)    -   W2=Peak width at baseline of NANA (min)

Resolution value must be ∃1.5.

The number of theoretical plates can be calculated using the followingequation:

N=16(T2/W2)²

-   -   Where:    -   N=Number of Theoretical Plates    -   T2=Retention time (min) of N-acetylneuraminic acid    -   W2=Peak width at baseline of N-acetylneuraminic acid (min)

Number of theoretical plates must be 2000. The CTLA4^(A29YL104E)-Ighydrolysis profile chromatagram should be similar to FIG. 48.

Samples were analyzed by Reverse Phase HPLC (RP-HPLC) in the followingorder: NANA and NGNA standards were first injected followed by injectionof samples, in duplicate if needed (for example, CTLA4^(A29YL104E)-Ig,etc). After sample analysis, the column was washed with Mobile Phase Bfor 20 minutes at 0.5 mL/minute. If needed, the column can be reversed.

Determination of the Molar Ratio (MR) of Sialic Acid (NANA or NGNA) toProtein.

The molar ratio of sialic acids to protein can be calculated byMillennium or Empower software.

Dilution factor:

$D = \frac{V_{protein} + V_{water}}{V_{protein}}$

where,

-   -   V_(protein)=volume of protein stock solution added (μl),    -   V_(water)=volume of water added (μl)

Protein working solution (protein used for hydrolysis) concentration(μM):

$C_{protein} = {\frac{C_{A\; 280}}{{MW}_{beletacept}} \times \frac{1}{D} \times 10^{6}}$

where,

-   -   C_(protein)=Concentration of the protein working solution (μM),    -   C_(A280)=A₂₈₀ concentration of the protein stock solution        (mg/ml),    -   MW_(CTLA4A29YL104E-Ig)=molecular weight of CTLA4A29YL104E-Ig,        91232 Da.    -   Concentration of Sialic Acids in the protein working solution        (μM):

$C_{unknown} = {C_{std} \times \frac{A_{u}}{A_{std}}}$

where,

-   -   C_(unknown)=Concentration of sialic acid (NANA or NGNA) in the        unknown sample    -   C_(std)=Concentration of sialic acid (NANA or NGNA) in the        standard (μM0    -   A_(u)=peak area of sialic acid (NANA or NGNA) in the unknown        sample    -   A_(std)=peak area of sialic acid (NANA or NGNA) in the standard

Molar ratio (M.R.) of sialic acid (NANA or NGNA) to protein:

${M.\; R.} = \frac{C_{unknown}}{C_{protein}}$

Calculation of total Molar ratio of sialic acid to protein (TSA):

TSA=NANA Molar Ratio+NGNA Molar Ratio

The relative standard deviation for the area counts of two bracketedNANA standards should be <10%. The relative standard deviation for thearea counts of two bracketed NGNA standards should be <10%. The relativestandard deviation for the area counts of two independent hydrolysatesshould be <10%.

In one embodiment, the average molar ratios for sialic acids in theCTLA4^(A29YL104E)-Ig the must be within the ranges specified in theTable directly below.

Molar Ratio Range of CTLA4^(A29YL104E)-Ig Material Monosaccharide RangeNANA 5.0-10.0 NGNA <1.5The % deviation of molar ratio for sialic acids in the referencematerial and samples for the two sample preparations must be ≤15%, ≤20%,≤25%, ≤30%, or ≤35%.

Example 40 An In-Vitro Cell Based Bioassay for CTLA4^(A29YL104E)-Ig

T cells require two signals for activation and subsequent proliferation.The first signal is provided by the interaction of an antigenic peptidewith the TCR-CD3 complex. The second co-stimulatory signal occurs withthe interaction between CD28 on the T cell and the B7 protein on anantigen-presenting cell. Upon receipt of these two signals, T cellssecrete the cytokine Interleukin 2 (IL-2). Release of IL-2 leads tocellular activation and proliferation. CTLA4^(A29YL104E)-Ig, a soluble,immunosuppressive compound, also binds to the B7 protein on the antigenpresenting cell, thus blocking functional interaction with CD28 andpreventing the co-stimulatory signal that is necessary for IL-2production.

In this method, Jurkat T cells transfected with the luciferase gene,under the control of the IL-2 promoter, are co-stimulated with Daudi Bcells in the presence of anti-CD3. The co-stimulation activates the IL-2promoter, which in turn produces luciferase protein. The resultingluminescent signal is measured using a Luciferase Assay System. In thissystem, CTLA4^(A29YL104E)-Ig produces a dose-dependent decrease inluciferase activity.

This method examines the effect of CTLA4^(A29YL104E)-Ig on theco-stimulatory signal needed for IL-2 production. The presence ofsoluble CTLA4^(A29YL104E)-Ig prevents signaling between the T cell andantigen-presenting cell. Without this signal, IL-2 is not produced, thuspreventing the clonal expansion of T cells. A vector with the luciferasegene was created using the IL-2 promoter. Jurkat T cells were thentransfected with this reporter vector. A positive clone, Jurkat.CA, wasselected and used in the method.

This bioassay involves stimulating transfected T cells (Jurkat.CA) withanti-CD3 and B cells (Daudi). Co-stimulation provided by the B cells isinhibited by the addition of CTLA4^(A29YL104E)-Ig. Jurkat.CA and Daudicells are seeded into the wells of a 96 well, white, opaque, flat-bottomplate and stimulated with anti-CD3 in the presence of differentconcentrations of CTLA4^(A29YL104E)-Ig. After a 16 to 20 hour incubationat 37° C., the wells are assayed for luciferase activity. Inhibition ofco-stimulation by CTLA4^(A29YL104E)-Ig is seen as a dose-dependentdecrease in luciferase activity. FIG. 93 is a procedure flow chart.

REAGENTS: Daudi Cell Culture Media (10% fetal bovine serum, 1% MEMsodium pyruvate in RPMI 1640); Jurkat.CA Cell Culture Media (10% calfserum, 1% MEM sodium pyruvate, 400 μg/mL of geneticin in RPMI 1640);Bioassay Media (0.2 μg/mL of anti-CD3 antibody and 1%penicillin-streptomycin solution in Daudi Cell Culture Media);Bright-Glo Luciferase Solution from assay system (Promega, Catalog #E2620).

INSTRUMENTATION: Nikon, Diaphot 200 Inverted microscope; PackardTopCount NXT Luminometer; Tecan Genesis Liquid Handler; Coulter Vi-CellCell Counter; Zymark RapidPlate-96.

Preparation of Working Solutions: 3 mL of CTLA4^(A29YL104E)-Ig solutions(5000 ng/mL) in bioassay media.

Daudi Cell Culture Media. Add 300 mL of RPMI 1640 to a 1 L Corningfilter unit. Then add 100 mL of fetal bovine serum and 10 mL of MEMsodium pyruvate. Add enough RPMI 1640 to make 1 liter. Filter and storeat 4° C. for up to one month.

Jurkat.CA Cell Culture Media. Add 300 mL of RPMI 1640 to a 1 L Corningfilter unit. Then add 100 mL of calf serum, 10 mL of MEM sodiumpyruvate, and 8 mL of geneticin at 50 mg/mL (final concentration is 400μg/mL). Add enough RPMI 1640 to make 1 liter. Filter and store at 4° C.for up to one month.

Bioassay Media. Add 100 mL of Daudi Cell Culture Media (2.1) to a 100 mLmedia bottle. Then add anti-CD3 antibody to a concentration of 0.2 μg/mLand 1 mL of penicillin-streptomycin solution (1.11). Mix gently byinversions and store at room temperature for no longer than 8 hours.

Bright-Glo Luciferase Solution. Prepare the solution, as described inthe system (1.21), by adding assay buffer to the luciferase substrateand mixing by inversion. The reagent should be used within 2 hours orstored at −20° C. and protected from light for up to 4 weeks.

Cell Line Maintenance. Determine the number of cells per mL for both theJurkat.CA and Daudi cells lines by counting with a cell counter. Cellsshould be between 1×10⁵ and 1.5×10⁶ cells/mL. Combine 12×10⁶ Jurkat.CAcells and 12×10⁶ Daudi cells in a sterile centrifuge tube. Centrifugethe cells at ˜125×g for 10 minutes at room temperature. Thoroughlyre-suspend the cells in 9 mL of Daudi cell culture media (2.1) by gentlypipetting repeatedly with a serological pipet until no cell clumps arevisible to give a concentration of 2.7×10⁶ cells/mL. Verify the cellconcentration by counting cells on a cell counter. Seed the re-suspendedcells into the wells of a 96 well plate (1.3) at 75 mL per well (200,000cells per well). Incubate the plate at incubator set points of 37° C.,5% CO₂, and 85% humidity while the standards, quality controls, andsamples are prepared. Preparation of the nominal concentrations ofCTLA4^(A29YL104E)-Ig for the standards, quality controls, and samples 1and 2. Prepare 3 mL of CTLA4^(A29YL104E)-Ig material working solution at5000 ng/mL in bioassay media (2.3) for use in the standard curve.Prepare 3 mL of CTLA4^(A29YL104E)-Ig material working solution at 5000ng/mL in bioassay media (2.3) for use in the quality control curve.Prepare 3 mL of each of the two CTLA4^(A29YL104E)-Ig Sample workingsolutions at 5000 ng/mL in bioassay media (2.3) for use in the samplecurves. (Approximate concentrations for CTLA4A29YL104E-Ig samples may beused to prepare the 8 point curves and relative potency values may becorrected as described in 5.5 when the determined concentration isavailable).

Eight point curves were prepared for the standard, quality control andsamples at the concentrations of 5000, 200, 100, 50, 25, 10, 5, and 0.1ng/mL CTLA4^(A29YL104E)-Ig as shown in Table 55 below for finalconcentrations in the assay, after twofold dilution into the plate, of2500, 100, 50, 25, 12.5, 5, 2.5, and 0.05 ng/mL.

TABLE 55 Dilutions used to generate standard curves. Curve PointStandard Curve Quality Control Sample 1 Sample 2 1 5000 ng/mL 5000 ng/mL5000 ng/mL 5000 ng/mL 2 200 200 200 200 3 100 100 100 100 4 50 50 50 505 25 25 25 25 6 10 10 10 10 7 5 5 5 5 8 0.1 0.1 0.1 0.1

Two plate maps may be used. The random plate map requires the use of aliquid handler for setup. The ordered plate map has adjacent triplicatesand each curve point for the test articles added in a sequential orderedlayout. For the random plate map, add 75 μL of each solution (4.8) tothe appropriate wells of the plate containing cells (4.5) as shown inthe plate map below. For the ordered plate map, add 75 μL of eachsolution (4.8) to the appropriate wells of two plates containing cells(4.5) as shown in the plate map below.

Seal the plate(s) with TopSeal-A (1.22). Ensure that the seal is tightlyin place. There should be no air gaps. Incubate the plate(s) atincubator set points of 37° C., 5% CO₂, and 85% humidity for 16 to 20hours. Equilibrate the plate(s) and Bright-Glo luciferase solution (2.4)to instrument temperature. Add 150 μL of Bright-Glo luciferase solutionto each well simultaneously and mix. Place the plate in the TopCount NXTimmediately after mixing to equilibrate in the dark for 10 minutes.Measure the luminescent signal in a TopCount NXT using a 1 secondintegration per well or as appropriate to the particular type ofluminometer used. The output from the TopCount NXT is recorded. Whenusing the ordered plate map, two plates will be read. The first plate(upright) will be read with well A1 in the upper left hand corner. Thesecond plate (inverted) will be read with well A1 in the lower righthand corner.

Bioassay Random Plate Map Setup

1 2 3 4 5 6 7 8 9 10 11 12 A QC Smp1 Stnd Stnd QC Stnd Stnd Smp2 Smp2Stnd Smp1 Stnd 0.05 12.5 0.05 2500 12.5 100 5 5 12.5 2.5 12.5 25 B StndSmp2 Smp1 Smp1 Smp1 QC Stnd QC Smp1 QC QC Smp2 5 25 5 0.05 12.5 2.5 0.052500 25 25 5 100 C Smp2 Stnd QC Stnd QC QC Smp2 Smp1 QC Smp1 QC Stnd 5100 5 2.5 0.05 5 100 0.05 100 100 50 5 D Smp1 Smp2 Smp1 QC Smp1 Smp2Smp1 Stnd Smp2 QC Stnd Smp1 100 12.5 50 50 2500 2500 2.5 25 5 2500 10050 E QC Smp2 QC Smp2 Smp2 Smp2 Stnd Smp1 Smp1 Smp1 Smp1 Smp2 100 100 252500 12.5 50 2500 25 5 2.5 0.05 2.5 F QC Stnd Smp1 Stnd Stnd Smp1 StndSmp2 Smp2 Stnd QC Smp2 2500 12.5 2.5 25 12.5 5 2.5 2.5 0.05 2500 2.5 50G QC Smp2 Smp2 Smp1 Smp2 QC Smp1 QC Stnd QC Smp1 Smp2 2.5 50 0.05 250.05 50 100 100 0.05 0.05 2500 2500 H Stnd Smp1 Smp2 QC QC Smp2 Smp1Stnd Stnd QC Smp2 Stnd 50 2500 2.5 12.5 25 25 50 50 50 12.5 25 12.5Stnd: Standard from 2500 to 0.05 ng/mL final concentration in the well.QC: Quality Control from 2500 to 0.05 ng/mL mL final concentration inthe well. Smp1, Smp2: Samples 1 to 2 from 2500 to 0.05 ng/mL mL finalconcentration in the well.

Bioassay Ordered Plate Map Setup:

1 2 3 4 5 6 7 8 9 10 11 12 A Stnd Stnd Stnd Stnd Stnd Stnd Stnd StndStnd Stnd Stnd Stnd 2500 2500 2500 100 100 100 50 50 50 25 25 25 B StndStnd Stnd Stnd Stnd Stnd Stnd Stnd Stnd Stnd Stnd Stnd 12.5 12.5 12.5 55 5 2.5 2.5 2.5 0.05 0.05 0.05 C QC QC QC QC QC QC QC QC QC QC QC QC2500 2500 2500 100 100 100 50 50 50 25 25 25 D QC QC QC QC QC QC QC QCQC QC QC QC 12.5 12.5 12.5 5 5 5 2.5 2.5 2.5 0.05 0.05 0.05 E Smp1 Smp1Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 2500 2500 2500 100 100100 50 50 50 25 25 25 F Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1 Smp1Smp1 Smp1 Smp1 12.5 12.5 12.5 5 5 5 2.5 2.5 2.5 0.05 0.05 0.05 G Smp2Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 2500 2500 2500100 100 100 50 50 50 25 25 25 H Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2 Smp2Smp2 Smp2 Smp2 Smp2 12.5 12.5 12.5 5 5 5 2.5 2.5 2.5 0.05 0.05 0.05Stnd: Standard from 2500 to 0.05 ng/mL mL final concentration in thewell. QC: Quality Control from 2500 to 0.05 ng/mL mL final concentrationin the well. Smp1, Smp2: Samples 1 to 2 from 2500 to 0.05 ng/mL mL finalconcentration in the well.

200,000 cells were added per well of a 96 well plate and were incubatedat 37° C., 5% CO₂, and 85% humidity. 12×10⁶ Jurkat.CA cells and 12×10⁶Daudi cells were combined in a sterile centrifuge tube. The cells werecentrifuged at ˜125×g for 10 minutes at room temperature and werethoroughly re-suspend in 9 mL of Daudi cell culture media by gentlypipetting repeatedly with a serological pipet until no cell clumps werevisible to give a concentration of 2.7×10⁶ cells/mL. 75 μL of eachsolution from Table 55 was added to the appropriate wells of the platecontaining cells The plate(s) were then sealed with TopSeal-A andincubated at 37° C., 5% CO₂, and 85% humidity for 16 to 20 hours. Afterthe plates and Bright-Glo luciferase solution were equilibrated to theinstrument temperature, 150 μL of Bright-Glo luciferase solution wasadded to each well simultaneously and were mixed. A plate is then placedin the TopCount NXT immediately after mixing for equilibration in thedark for 10 minutes. The luminescent signal was then measured in aTopCount NXT using a 1 second integration per well or as appropriate tothe particular type of luminometer used.

The output from the TopCount NXT was recorded, read into a standardanlysis program, and data were transformed by taking their logarithm(base 10). The transformed data from each article were fit to afour-parameter logistic model as shown in the equation below:

$\begin{matrix}{{{Log}_{10}\left( y_{jk} \right)} = {D + \frac{\left( {A - D} \right)}{1 + \left( \frac{x_{j}}{C} \right)^{B}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

-   -   Where:    -   A is the top plateau of the curve, D is the bottom plateau of        the curve, B is the slope factor, and C is the concentration        that produces an effect equal to the average of A and D.

An R² statistic, and a lack-of-fit F-test can be calculated for eacharticle. A ratio of the minimum, maximum and slope of the test articlesrelative to the standard material can also be calculated. In addition,confidence intervals for the ratios can also be computed.

The relative potency of each article was determined by fitting a singleequation to the data from the article of interest combined with the datafrom the reference article.

${{Log}_{10}\left( y_{{ijk}\;} \right)} = {D + \frac{\left( {A - D} \right)}{1 + \left( \frac{x_{i\; j}}{C_{A}*\left( \frac{C_{R}}{C_{A}} \right)^{I}} \right)^{B}}}$

-   -   Where:    -   A, B and D parameters are common to both the reference and test        article and C_(R) is the reference parameter, C_(A) is the test        article parameter, and the ratio C_(R)/C_(A) is the relative        potency. The superscript I is an indicator variable. It is set        equal to 1 if the data come from the article of interest, and 0        if the data come from the standard material.

The relative potency of each test article was translated to a percentagescale and the relative potency was given as output from the program.

Adjustment of Relative Potency Values Obtained With ApproximateConcentrations: Due to the time lag between sample receipt and obtaininga precise protein concentration, a sample may be tested in the assay atan approximate concentration and results adjusted when the preciseconcentration is determined. This adjustment is performed using Equation2 below where the relative potency determined in the assay is multipliedby the ratio of the CTLA4^(A29YL104E)-Ig concentration used to set upthe assay to the determined CTLA4^(A29YL104E)-Ig sample concentration.

$\begin{matrix}{{{Reportable}\mspace{20mu} {Relative}\mspace{20mu} {Potency}} = \frac{{Observed}\mspace{14mu} {Relative}\mspace{14mu} {Potency}^{*}{Concentration}\mspace{20mu} {Used}}{{Determined}\mspace{14mu} {Concentration}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

-   -   Example:    -   Sample was tested at a protein concentration of 25 mg/mL in the        assay.    -   Relative Potency determined was 105%.    -   Determined CTLA4^(A29YL104E)-Ig concentration was determined to        be 25.5 mg/mL    -   Reportable Relative Potency=(105*25)/25.5=103%.

Standard Material: The EC₅₀ value for the Standard of the output shouldbe between 5 and 35 ng/mL. The difference between the 2500 ng/mL and the0.05 ng/mL concentration standards (range) should be ≥40,000 counts persecond (CPS). The R-squared value for the reference should be greaterthan 0.95.

The test article relative potency values in Table 9 must be between 25and 175% of the reference standard, which is the range of the assay. Ifthe relative potency values are outside this range, then the sample mustbe diluted or concentrated in order to fall within this range and thesample reanalyzed.

Daudi B Cell Line:

Source: Daudi cells were obtained from ATCC. A master bank was generatedcomprised of 64 vials. A working bank was created from a master bankvial after 4 passages. (NOTE: Passage 0 is considered the thaw and then3 additional passages were made prior to working bank generation).

Media: Daudi cells are grown in RPMI 1640 medium (containing HEPES andL-glutamine) supplemented with 10% fetal bovine serum and 1% sodiumpyruvate.

Incubator Conditions: Cells are maintained in a vented T-flask at 37°C., 5% CO₂, and 70-90% humidity.

Thawing Protocol: A vial of cells is removed from a liquid nitrogenfreezer and thawed in a 37° C. water bath. The contents are mixed with10 mL of culture medium. Cells are counted and then collected bycentrifugation at 125×g for 10 minutes. Following centrifugation, thesupernatant is removed and the cells are suspended in fresh media at3×10⁵ viable cells/mL. The cells are defined to be at passage 0 at thispoint.

Growth Properties: The cell line grows in suspension.

Subculturing: Cultures are maintained by passage twice a week with nolonger than 5 days between passages. Cells are passaged in a ventedT-flask with fresh medium between 0.5×10⁵ and 2×10⁵ viable cells/mL.Cells should not reach a density greater than 1.5×10⁶ cells/mL. Cellsshould be greater than 80% viable as assessed by trypan blue staining.The date and passage number should be labeled on the T-flask afterpassage.

Doubling Times: The doubling time ranges from 18 to 26 hours.

Passage Limitations: The cells from a working bank should be passed 3times before using in the bioassay i.e. they should be at passage 3 orhigher. The cells should only remain in culture for 20 passages. A newworking vial should be thawed at that time.

Freezing Protocol: Cells are frozen from 5 to 10×10⁶ cells/mL in acryovial. Cryoprotectant medium is prepared by supplementing completeculture medium with 5% (v/v) DMSO. The cells are frozen at a rate of 1°C./minute until they reach liquid nitrogen temperature (˜190° C.).

Jurkat.CA T Cell Line:

Source: Jurkat T cells were transfected with a plasmid encoding aCTLA4-Ig molecule. A working bank was created from a master bank vialafter 3 passages (NOTE: Passage 0 is considered the thaw and then 2additional passages were made prior to working bank generation).

Media: Jurkat.CA cells are grown in RPMI 1640 medium (containing HEPESand L-glutamine supplemented with 10% calf serum and 1% sodium pyruvatesupplemented with geneticin (G418 sulfate) at a final concentration of400 μg/mL.

Incubator Conditions: Cells are maintained in a vented T flask at 37°C., 5% CO₂, and 70-90% humidity.

Thawing Protocol: A vial of cells is removed from a liquid nitrogenfreezer and thawed in a 37° C. water bath. The contents are mixed with10 mL of culture medium. Cells are counted and then collected bycentrifugation at 125×g for 10 minutes. Following centrifugation, thesupernatant is removed and the cells are suspended in fresh media at3×10⁵ viable cells/mL. The cells are defined to be at passage 0 at thispoint.

Growth Properties: The cell line grows in suspension.

Subculturing: Cultures are maintained by passage twice a week with nolonger than 5 days between passages. Cells are passaged in a ventedT-flask with fresh medium between 0.5 and 2×10⁵ viable cells/mL. Cellsshould not reach a density greater than 1.5×10⁶ cells/mL. Cells shouldbe greater than 80% viable as assessed by trypan blue staining. The dateand passage number should be labeled on the T-flask after passage.

Doubling Times: The doubling time ranges from 18 to 26 hours.

Passage Limitations: The cells from a working bank should be passed 3times before using in the bioassay i.e. they should be at passage 3 orhigher. The cells should only remain in culture for 20 passages. A newworking vial should be thawed at that time. [001301] Freezing Protocol:Cells are frozen from 5 to 10×10⁶ cells/mL in a cryovial. Cryoprotectantmedium is prepared by supplementing complete culture medium with 5%(v/v) DMSO. The cells are frozen at a rate of 1° C/minute until theyreach liquid nitrogen temperature (˜190° C.).

Example 41 Determination of Bio-Specific Binding of CTLA4^(A29YL104E)-Igto the B7.1-Ig Receptor by Surface Plasmon Resonance (BIAcore)

The relative binding of CTLA4^(A29YL104E)-Ig samples to the B7.1Igreceptor was measured by surface plasmon resonance using the BIAcoreinstrument. In this assay CTLA4^(A29YL104E)-Ig binds to a B7.1Igimmunoglobulin fusion protein derived from the APC cell membrane proteinB7.1. After immobilizing the B7.1Ig receptor to a high density on thesurface of an activated sensor chip, CTLA4^(A29YL104E)-Ig material,quality controls, and samples are diluted to generate bindingsensorgrams. The initial binding rate (slope)/Resonance Units (RU) boundof CTLA4^(A29YL104E)-Ig to immobilized B7.1Ig surface is measured underthe mass transfer (diffusion) limited conditions on this high densityB7.1Ig surface. The initial binding rate in resonance units per second(RU's) correlates directly with the bioactive concentration. The bindingrates of samples are calculated into an active concentration using thereference standard curve where the binding rate of aCTLA4^(A29YL104E)-Ig material is plotted against the concentration. Thefinal results are either expressed by percent binding of sample relativeto CTLA4^(A29YL104E)-Ig material or as a concentration. A method outlineis shown in FIG. 94.

REAGENTS: Amine Coupling Kit BIA Certified (kit contains one vial each:115 mg N-hydroxysuccinimide (NHS), 750 mg N-ethyl-N′-(3-dimethyl) (EDC),and ethanolamine); Regeneration Buffer (10 mM Sodium Citrate, 100 mMNaCl, pH 4.0);

INSTRUMENTATION: BIAcore C Instrument with a PC compatible computer(BIAcore (Catalot No.BR-1100-51)); BIAcore C Control Software version1.0.2; BIAcore Evaluation Software 1.0; BIAcore 96-well microtiter plateU-shape BIAcore (Catalog No. BR-1005-03); BIAcore 96-well micorplateFoils plate sealer (Catalog No. BR-1005-78).

Materials

Sensor Chip CM5, certified grade BIAcore AB (Catalog No. BR-1000-12)BIACORE ® C Instrument Handbook BIAcore (Catalog No. BR-1005-11) HBSBuffer BIA certified 10 mM HEPES BIAcore (Catalog No. BR1001-88) pH 7.4,150 mM NaCl, 3.4 mM EDTA 0.005% v/v Surfactant P20 BIAnormalizingSolution (40% glycerol BIAcore AB (Catalog No. BR-1002-22) solution)Amine Coupling Kit BIA certified 115 mg BIAcore (Catalog No. BR-1000-50)N-hydroxysuccinimide 750 mg N-ethyl- N′-(3-dimethyl) Sodium Chloride(NaCl) Sigma (Catalog No. S-9888) Acetate Buffer pH 5.0 BIAcore (CatalogNo. BR-1003-51) Sodium Citrate (C₆H₃Na₃O) Sigma (Catalog No. S-4641)Hydrochloric Acid (HCl) Fisher Scientific (Catalog No. A144-212) BIAcoreMaintenance Kit BIAcore (Catalog No. BR-1006-67)

Reagents

Amine Coupling Kit BIA Certified. The kit contains one vial each: 115 mgN-hydroxysuccinimide, 750 mg N-ethyl-N′-(3-dimethyl) and ethanolamine.Aliquot each solution according to manufacturer's directions and storeas indicated below:

Store aliquots of 11.5 mg/ml N-hydroxysuccinimide (NHS) at −20° C. Thisfrozen aliquot expires 2 months from the date of preparation. Storealiquots of 75 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC) at −20° C. This frozen aliquot expires 2 months fromthe date of preparation. Store aliquots of 1.0 M ethanolamine-HCl pH 8.5at −20° C. This frozen aliquot expires 2 months from the date ofpreparation. Regeneration Buffer (10 mM Sodium Citrate, 100 mM NaCl, pH4.0). Analytically weigh out 1.5±0.1 g Sodium Citrate and 2.9±0.1 gNaCl. Add to 500 mL Milli-Q Water and adjust pH to 4.0 with 1 N HCl.Filter the solution with a 0.22 μm filter then aliquot the 500 mL into45 mL/50 mL conical tubes. Solution expires 6 months from the date ofpreparation when stored at 2-8° C.

Instrumentation

BIAcore C Instrument with a PC BIAcore (Catalot No. BR-1100-51)compatible computer BIAcore C Control Software: BIAcore, as providedwith BIAcore C instrument, version 1.0.2 Evaluation Software BIAcore, asprovided with BIAcoreC instrument, version 1.0 pH Meter ORION, Model720A+

PREPARATION OF SENSOR CHIP. Insert a new Sensor Chip CMS into thecassette port on the detector unit of the instrument. Prime the systemas described in the BIAcore C Handbook using HBS-EP buffer.

OPERATION OF BIAcore C INSTRUMENT FOR METHOD FUNCTION. The BIAcore Cinstrument is controlled from a compatible PC computer under MicrosoftWindows environment with the BIAcore C Control Software. Refer to theWork Instructions, “Operation of BIAcore C Instrument” and “Calibrationand Maintenance of BIAcore C Instrument” for use and maintenance ofBIAcore C instrument.

Immobilization Method of B7.1Ig

Preparation of B7.1Ig: A vial containing B7.1Ig was thawed at 22.5±5° C.and diluted to a concentration that immobilizes 3000-9000 RU, using 10mM Acetate pH 5.0 buffer as diluent. A vial of EDC, NHS, andEthanolamine were each thawed from the Amine Coupling Kit as described.Reagent and ligand vials were then placed in sample rack as instructedby the BIAcore program, and immobilization was initiated according toBIAcore instrument instructions.

Preparation of CTLA4^(A29YL104E)-Ig Standard Curve

Standards and samples (such as CTLA4^(A29YL104E)-Ig, etc) were thawed atroom temperature. Standards used to generate a standard curve werediluted to the target concentrations of the standard curve (250, 500,1000, 2000, 4000, and 8000 ng/mL).

For the dilution of a standards sample (such as a CTLA4^(A29YL104E)-Igmaterial stock) and the concentration was 25 mg/mL, one could performthe following dilutions:

-   -   i. Dilute 1/50 (500 μg/mL) by adding 20 μL to 980 μL HBS-EP    -   ii. Dilute to 16 μg/mL by adding 30 μL of 500 μg/mL to 908 μL        HBS-EP    -   iii. Serially dilute in 1/2 steps (500 μL of previous        dilution+500 μL HBS-EP) down to 250 ng/mL

Each standard can be analyzed in duplicate injections that will resultin 2 slope values.

QUALITY CONTROL SAMPLES. Quality Control (QC) samples ofCTLA4^(A29YL104E)-Ig are prepared in HBS-EP buffer at the three targetconcentration levels of 750, 2500, and 5000 ng/mL and are frozen in 7 mmplastic vials as 200 μL aliquots at −80° C. The CTLA4^(A29YL104E)-Igconcentrations in the QC samples are determined in three independentconcentration analysis experiments using this method. In each experimentall QC sample injections must be acceptable, detecting within±20% ofnominal concentrations. The qualified and frozen QC samples expire 6months after preparation. On the day of an experiment, thaw one vialeach of the three QC samples at room temperature. Place QC samples inthe sample rack positions as described in the method wizard and analyzeeach QC with triplicate injections. Alternatively, QCs can be preparedfresh on the day of analysis.

CTLA4^(A29YL104E)-Ig TEST SAMPLES. CTLA4^(A29YL104E)-Ig samples have tobe diluted to a concentration within the range of the assay (between 750and 5000 ng/mL). Sample with a known approximate CTLA4^(A29YL104E)-Igconcentration should be diluted to a target concentration of 2000 ng/mL.After thawing samples at room temperature they are diluted to a targetconcentration of 2000 ng/mL using HBS-EP buffer. Sample dilutions can beprepared for analyses in either BIAcore certified polypropylene testtubes or a 96-well microtiter plate. The following is an example for thedilution of a CTLA4^(A29YL104E)-test sample:

After thawing samples at room temperature, CTLA4^(A29YL104E)-Ig sampleswere diluted to a concentration within the range of the assay (such as,between 750 ng/ml and 5000 ng/mL). Samples with known approximateCTLA4^(A29YL104E)-Ig concentration should be diluted to a targetconcentration of 2000 ng/mL using HBS-EP buffer in either BIAcorecertified polypropylene test tubes or a 96-well microtiter plate.

The following was an example for the dilution of a CTLA4^(A29YL104E)-Igsample (concentration=25.0 mg/mL):

-   -   i. Dilute 1/50 (500 μg/mL) by adding 20 μL to 980 μL HBS-EP    -   ii. Dilute 1/25 (20 μg/mL) by adding 30 μL of 500 μg/mL to 720        μL HBS-EP    -   iii. Dilute 1/10 (2 μg/mL) by adding 100 μL of 20 μg/mL to 900        μL HBS-EP

Vortex each dilution at moderate speed for 2-4 seconds. Samples areprepared in triplicate (three independent dilutions from the stocksample solution), and each dilution is analyzed with one injection.Samples were prepared in triplicate (three independent dilutions fromthe stock sample solution), and each dilution was analyzed with oneinjection.

STARTING ANALYSIS USING THE BIACORE C SOFTWARE: Open BIAcore C ControlSoftware and select File->Project->Published->8164-2 ->ConcentrationAnalysis Wizard, Click on “Next” and select a published templateappropriate for the analysis to be performed, for example“CTLA4^(A29YL104E)-Ig Sample Concentration Analysis. blw”. Enter themaximum number of samples for the planned analysis. This number ofsamples includes replicates, so, for example, to analyze 8 samples intriplicate the number 24 should be entered. Select Flow Cell (1-4) onwhich the B7.1Ig ligand was immobilized. Click on “Next” and entersample ID's, dilution factors, and number of replicates. Click on “Next”and check vial positions. At this point the positions of vials can bemoved to the desired locations. Click on “Next” and confirm the set-upand choices for the assay on the “Preparation for Analysis” screen.Click on “Next” on to scroll down. Place the standards, QC's,regeneration solutions, and test samples in the appropriate positions.Click on “Start” to start the analysis.

DATA EVALUATION. Start BIAcore C Software and open the BIAcorefile***.blr that was generated. Then select “Wizard Results” from the“View” menu to evaluate the data.

Exemplary Values for B7.1Ig Surface

The mass of B7.1Ig immobilized on an activated flow cell was expressedas RU's (resonance units). The surface mass should be between 3000 to9000 RU's for optimal assay performance. If the surface mass does notmeet this exemplary value, an adjustment of the activation time (EDC/NHSinjection) or the B7.1Ig concentration can be made and another flow cellcan be immobilized.

Exemplary Values for Baseline Drift

The baseline drift of each run was calculated as the percent change ofthe baseline (“absolute response values”) between each cycle relative tothe immobilized surface mass of the ligand. The largest percent changebetween any two cycles of the run must be ≤5.0%. For example: ifBaseline at cycle 20=13500 RU, Baseline at cycle 23=13650 RU, and B7.1Igsurface density=6000 RU, then Baselinedrift=(13650-13500)/6000×100=2.5%.

Exemplary Values for the Standards

Exemplary values apply to the standard curve concentrations at or above500 ng/mL. The coefficient of variation (% CV) of the values at eachslope values at each standard concentration used to determine thestandard curve must be within±10%. The mean calculated concentrationvalues (ng/mL) at each standard concentration must be within 15% of thetarget (nominal) value. The difference between the calculatedconcentration and the target (nominal concentration) can be divided bythe target (nominal) concentration and multiplied by 100. For example,at standard 500 ng/mL, the BIAcore C calculated concentration is 510ng/mL. The % deviation will be calculated as follows: (510 ng/mL-500ng/mL)/500 ng/mL×100%=2.0%.

Exemplary values for QC Samples: The % CV of the triplicate calculatedconcentration values for each QC sample target concentration must bewithin±10%. QC sample results must be within±15% of their respectivetarget values for at least seven of the nine QC determinations. Thedifference in slope values between the first and last QC injection atthe 2500 ng/mL level must be within±5.0%. For example, the first slopevalue is 10.366 RU/s and the last slope vales is 10.230 RU/s, thepercent difference will be as following:

(10.366−10.230)/10.366×100%=1.3%

Exemplary Values for Test Samples

The % CV of the triplicate observations obtained for each test sampletarget concentration must be within 20%. The slope values for a samplemust be in the range of the method: average slope at quality controlsample 1≤average slope of sample≤average slope of quality control sample3.

Sample Results Calculations

To determine the percent binding of a test sample relative to theCTLA4^(A29YL104E)-Ig material, the concentration value for each testsample is calculated from the standard curve in ng/mL. The ResultsWizard of the BIAcore instrumentation calculates theCTLA4^(A29YL104E)-Ig concentration in the undiluted sample in mg/mLbased on the dilution factor entered for each sample.

Determining the percent binding: The calculated mean concentration valuefor each sample tested can be multiplied by 100 and divided by thereported protein concentration of the sample as determined by absorbancemeasured at 280 nm (A₂₈₀). The value can then be reported to the nearestwhole number as percent binding relative to standard samples. Forexample, the sample was diluted by a dilution factor of 12,500 (sampleprotein concentration of approximately 25 mg/mL). The calculated averageCTLA4^(A29YL104E)-Ig concentration from the triplicate injections of thesample is 25.3 mg/mL. The A₂₈₀ for the sample is 24.2 mg/mL. Thecalculated percent binding relative to standard samples can be asfollows: 25.3 mg/mL/24.2 mg/mL×100%=104.545%.

CTLA4^(A29YL104E)-Ig Immobilization Wizard Template

Immobilization Injection Parameters EDC/NHS B7.1-Ig Ethanolamine CitrateContact Time (min) 7 7 6 2 Flow Rate (μL/min) 5 5 5 10 Injection Volume(μL) 35 35 30 20

Immobilization Report Points Start of/ Time Before/ End Relative WindowID (s) After of Injection Type to (s) Report Baseline 10 Before Start1^(st) Inj AbsResp — 5 No of Activation 10 Before Start 2^(nd) InjRelResp Baseline 5 No of Immobilization 85 After End 3^(rd) Inj RelRespBaseline 5 Yes Level of

Example 42 Correlations between Carbohydrate Analytical Data of CTLA4-Igand Pharmacokinetic Data

The carbohydrate structures on CTLA4-Ig play an important role in thepharmacokinetics (PK) of the CTLA4-Ig therapeutic composition. Severalanalytical methods have been developed to characterize thesecarbohydrate structures. Two analytical parameters correlate well withclearance rates: the sialic acid (NANA) to CTLA4-Ig protein ratio andthe percent Domain III and IV from the carbohydrate profile. A thirdparameter (the galactose to mannose ratio from the monosaccharideanalysis) also appears to correlate well. For example, thespecifications for these parameters can be:

NANA:CTLA4-Ig Protein Ratio ≥8.0 Carbohydrate Profile Domains III and IV≥ 25% (Method 1) Galactose:Mannose Ratio ≥0.65

An important step in the CTLA4-Ig fermentation process can be theselection of harvest parameters that will maximize yield (titer) of thefinal product comprising characteristics specified herein (see Table 6).One of the parameters (NANA:protein ratio) is sufficiently rapid andaccurate to be used as one of the harvest parameters. A target molarratio of NANA to CTLA4-Ig protein of about 8.0 can ensure that mostharvests will have a NANA ratio >7.0. Enhancements during purificationcan subsequently produce CTLA4-Ig molecules with a NANA ratio >9.0.

CTLA4-Ig is a glycoprotein with several N-linked and O-linkedglycosylation sites. The N-linked carbohydrate structures are typicallybi-or tri-antennary and, if fully sialylated, terminate with thecarbohydrates NANA-Gal-GlcNAc. Most molecules are only partiallysialylated and contain some carbohydrate chains terminating with Gal orGlcNAc. The absence of terminal sialic acid (NANA) residues is a factorleading to increased exposure rates from the blood stream. In thisexample, data is presented indicating that terminal galactose alsoprovides some protection from rapid clearance.

A primary parameter used to evaluate glycosylation is the NANA:CTLA4-Igprotein molar ratio. Monkey PK studies had shown that acceptableclearance could be achieved using CTLA4-Ig molecules with a NANA ratioof 6.9. The first lot of Process Y CTLA4-Ig material (CTLA4-Igcomposition obtained from the Y Process) tested in monkeys had a NANAratio of 7.1. Surprisingly, it was found to clear twice as rapidly asCTLA4-Ig material with a NANA ratio of 6.9. A review of themonosaccharide analysis of the two samples indicated that the X Processmaterial had significantly more galactose than the CD-CHO processmaterial. A process was developed which included galactose in the feed(CD-CHO1). The CTLA4-Ig produced by this process had higher levels ofboth galactose and sialic acid than the Y Process material. Theanalytical and PK data for material from the CD-CHO1 process isdiscussed below and compared with the Process Y and Process X material.

Analytical Methods

The carbohydrate profile assay consists of the enzymatic removal of theentire carbohydrate structures and their separation by anion exchangechromatography. The carbohydrate peaks resolve into four or fiveclusters (“domains”) based largely upon the number of sialic acidresidues in each structure. Domain I is largely asialylated, Domain IIis monosialylated, Domain III is di-sialylated, Domain IV istri-sialylated and Domain V is tetra-sialyated. The area under each peakor domain can be determined and reported as a percentage of the totalarea under all the peaks.

The clearance rate was determined by injecting monkeys in triplicatewith 10 mg/kg of CTLA4-Ig, then following the decrease in serumconcentration over a 28-day period. The clearance rate is related to themeasured “area under the curve” or AUC. The AUC is related to clearance,a higher clearance rate is related to a smaller AUC and a lowerclearance rate to a larger AUC value. Higher values represent slowerclearance of CTLA4-Ig.

FIG. 49 depicts several of the many N-linked carbohydrate structuresfound in mammalian proteins. All chains share a common core structurecontaining two GlcNAc and three mannose residues. From this core, two tofour chains extend out consisting of one of three structures:-GlcNAc-Gal-NANA, -GlcNAc-Gal, or -GlcNAc (FIG. 49, structures (1), (2),and (3)). Assuming that the terminal sialic acid (NANA) is responsiblefor reducing clearance of the protein from the bloodstream. It came as asurprise that the clearance rate of CTLA4-Ig doubled in a monkey PKstudy (Table 56) even though the NANA ratio remained the same (Table56—samples CTLA4-Ig S1 and CTLA4-Ig (−) Gal, respectively, in FIGS.50-51). One difference observed between the samples, prepared indifferent media, was the galactose-to-protein molar ratio, which wassignificantly higher in the sample prepared by the Process X than byProcess Y (CTLA4-Ig). This suggested that the clearance rate wasprimarily determined by terminal GlcNAc residues, and that terminalgalactose might provide some protection. To increase the galactosylationof CTLA4-Ig, galactose was added to the feed for the Y Process. The newprocess (CD-CHO1; CTLA4-Ig S2 in FIGS. 50-51) significantly increasedboth the galactose and NANA molar ratios of the CTLA4-Ig in thebioreactors.

Further monkey PK studies have been carried out. These have includedCTLA4-Ig product produced by the three different processes (Process X,Process Y, and the CD-CHO1 Process; CTLA4-Ig S1, CTLA4-Ig (-) Gal, andCTLA4-Ig S2 respectively in FIGS. 50-51). In addition, CTLA4-Ig with avery low NANA ratio (recovered from a wash step in the purificationprocedure; see FIG. 61) has been tested. The analytical and PK data fromall of the samples tested to date are compiled in Table 56. In the mostrecent PK study (Table 56), CTLA4-Ig produced from an extended Process Yfermentation run (CTLA4-Ig S3) was evaluated.

FIG. 50 shows the correlation between the NANA ratio and the monkey PKAUC values for all of the samples in the four studies. Samples preparedby the Y Process (see CTLA4-Ig (−) Gal in FIG. 50) and the CD-CHO1Process (CTLA4-Ig S2 in FIG. 50) showed a strong correlation betweenthese parameters indicating that the NANA ratio has a significant impacton the clearance rate of CTLA4Ig. Analysis of the trendline for thesepoints indicates that at NANA=9, CTLA4-Ig prepared by the Y (CTLA4-Ig(−) Gal) or CD-CHO1 (CTLA4-Ig S2) process will clear at about the samerate as the Process X material CTLA4-Ig S1). Reducing the NANA ratio to8 reduces the AUC in the monkey PK by about 25%, while increasing theratio to 10 increases the AUC by about 25%. Although there is a strongcorrelation between NANA and AUC within the context of the Y (CTLA4-Ig(−) Gal) and CD-CHO1 (CTLA4-Ig S2) processes, it is important toremember that NANA=7 for X material (CTLA4-Ig S1) with the sameclearance rate as CD-CHO1 material (CTLA4-Ig S2) with a NANA ratio of 9.Therefore, the NANA ratio is not solely responsible for determining theclearance rate of CTLA4-Ig.

TABLE 56 Carbohydrate Evaluation of CTLA4-Ig. Ferm. Process X Process YProcess Y Process ASSAY PARAMETER MEAN SD MEAN SD MEAN SD Sialic AcidNaNA:PROT. 6.9 7.1 6.9 Carbohydrate % Domain 1 (PA) Profile % Domain 2(PA) % Domain 3 (PA) % Domain 4 (PA) % Domain 1 (M-1) 42.4% 49.1% 43.1%% Domain 2 (M-1) 28.2% 31.1% 32.8% % Domain 3 (M-1) 21.1% 15.9% 19.7% %Domain 4 (M-1) 7.4% 3.8% 3.7% Mono- Mannose 17.3 17.2 15.0 saccharideFucose 5.7 5.7 6.7 Analysis Galactose 13.1 8.1 9.1 GalNAc 2.7 3.4 3.2GlcNAc 19.9 21.3 26.3 Gal:Man 75.7% 47.1% 60.7% Gal:GlcNAc 65.8% 38.0%34.6% Monkey PK AUC (hrs * μg/ml 17060 1171 8832 2203 DS02051-1 MonkeyPK AUC (hrs * μg/ml) 15753 4395 7765 1247 DS02051-2 Monkey PK AUC (Hrs *μg/ml) 15459 DS02051-3 Monkey PK AUC (Hrs * μg/ml) DS03228 Ferm. ProcessY Process CD-CH01 ASSAY PARAMETER MEAN SD MEAN SD Sialic Acid NaNA:PROT.(M-2) 7.3 9.9 Carbohydrate % Domain 1 (PA) 36.0% Profile % Domain 2 (PA)35.6% % Domain 3 (PA) 23.1% % Domain 4 (PA) 5.2% % Domain 1 (M-1) 42.9%33.6% % Domain 2 (M-1) 33.5% 31.6% % Domain 3 (M-1) 18.4% 27.5% % Domain4 (M-1) 3.6% 5.9% Mono- Mannose 19.6 18.0 saccharide Fucose 4.6 4.8Analysis Galactose 11.1 15.8 GalNAc 3.3 3.3 GlcNAc 21.9 22.3 Gal:Man56.6% 87.8% Gal:GlcNAc 50.7% 70.9% Monkey PK AUC (hrs * μg/ml DS02051-1Monkey PK AUC (hrs * μg/ml) 7266 787 20445 2425 DS02051-2 Monkey PK AUC(Hrs * μg/ml) DS02051-3 Monkey PK AUC (Hrs * μg/ml) DS03228 (d12) (d16)Ferm. Process Process X CD-CH01 CD-CHO1 ASSAY PARAMETER MEAN SD MEAN SDMEAN SD Sialic Acid NaNA:PROT. 2.3 9.8 8.8 Carbohydrate % Domain 1 (PA)63.8% 34.1% 42.0% Profile % Domain 2 (PA) 26.5% 28.3% 30.1% % Domain 3(PA) 7.3% 25.7% 22.4% % Domain 4 (PA) 2.4% 10.7% 5.3% % Domain 1 (M-1)34.8% 38.2% % Domain 2 (M-1) 28.7% 34.8% % Domain 3 (M-1) 30.1% 22.5% %Domain 4 (M-1) 6.1% 4.3% % Domain 1 (M-2) 37.8% 44.4% % Domain 2 (M-2)34.5% 32.8% % Domain 3 (M-2) 22.8% 19.6% % Domain 4 (M-2) 5.0% 3.2%Mono- Mannose 17.9 13.6 14.5 saccharide Fucose 5.5 5.4 5.3 AnalysisGalactose 4.1 12.8 11.4 GalNAc 2.9 2.1 2.2 GlcNAc 22.0 26.3 19.9 Gal:Man22.9% 94.1% 78.6% Gal:GlcNAc 18.6% 48.7% 57.3% Monkey PK AUC (hrs *μg/ml DS02051-1 Monkey PK AUC (hrs * μg/ml) 2337 414 DS02051-2 Monkey PKAUC (Hrs * μg/ml) 20707 15779 DS02051-3 Monkey PK AUC (Hrs * μg/ml)DS03228 (d16) (d14) (d16) CTLA4-Ig S3 Ferm. Process Process X CD-CHO1CD-CHO1 ASSAY PARAMETER MEAN SD MEAN SD MEAN SD Sialic Acid NaNA:PROT.(BAS) 10.0 10.3 7.9 Carbohydrate % Domain 1 (PA) 38.6% 41.3% 56.2%Profile % Domain 2 (PA) 30.1% 32.2% 26.9% % Domain 3 (PA) 23.1% 21.9%13.1% % Domain 4 (PA) 7.6% 4.6% 3.7% % Domain 1 (M-1) 39.5% 49.0% %Domain 2 (M-1) 31.8% 29.1% % Domain 3 (M-1) 21.9% 15.7% % Domain 4 (M-1)7.8% 6.3% % Domain 1 (M-2) 38.2% 36.2% 47.1% % Domain 2 (M-2) 33.7%34.3% 33.6% % Domain 3 (M-2) 24.6% 21.1% 14.5% % Domain 4 (M-2) 3.6%8.4% 4.8% Mono- Mannose 14.8 14.7 11.8 saccharide Fucose 5.3 4.9 5.7Analysis Galactose 13.0 12.6 11.5 GalNAc 2.2 2.2 2.3 GlcNAc 20.2 16.328.4 Gal:Man 87.8% 85.7% 97.5% Gal:GlcNAc 64.4% 77.3% 40.5% Monkey PKAUC (hrs * μg/ml DS02051-1 Monkey PK AUC (hrs * μg/ml) DS02051-2 MonkeyPK AUC (Hrs * μg/ml) 18750 DS02051-3 Monkey PK AUC (Hrs * μg/ml) 177392546 9425 1504 DS03228 M-1 indicates that the CTLA4-Ig material wasanalyzed using Method 1, M-2 indicates that the CTLA4-Ig material wasanalyzed using a slightly different, Method 2. “PA” indicates materialthat is a Protein A - purified sample from fermentation broth.

Another monkey PK study (Table 56) was performed to compare clearancerates of CTLA4Ig produced by the three different processes (Process X,Process Y, and CD-CHO1 Process). In addition, CTLA4Ig with a very lowNANA ratio (recovered from a wash step in the purification procedure(PA)) was included in the study. CTLA4Ig prepared by the CD-CHO processin either SOL or 5000 L bioreactors had NANA molar ratios close to the XProcess material, and in the PK study, both had AUC values of about halfthe Process X value. CTLA4Ig produced by the CD-CHO1 process had ahigher NANA ratio than the Process X material (9.9 vs. 6.9) and an AUCvalue about 30% higher, indicating a slower clearance rate. The poorlysialylated and galactosylated wash material (NANA=2.3) cleared extremelyquickly (AUC=2337 hr-μg/ml vs. 15753 for the Process X material).

Another monkey PK study (Table 56) compared CTLA4Ig prepared by theCD-CHO1 process in 5,000 L bioreactors and harvested on different days.During the course of a fermentation run, the NANA ratio typically peaksat about Day 8, then gradually declines. From two runs, aliquots wereremoved on Days 12, 14, and 16, then purified. After purification, NANAratios ranged from 8.8 to 10.0. Day 12 and 14 samples (NANA=9.8 and 10.0respectively) had AUC values 20-30% higher than the Process X material.The Day 16 sample (NANA=8.8) had an AUC value almost identical to theProcess X material.

Another analytical tool to evaluate the glycosylation of CTLA4-Ig is thecarbohydrate profile. Entire N-linked carbohydrate structures areenzymatically removed and separated by anion exchange HPLC. A largenumber of peaks are generated which resolve into four or five domains(see FIGS. 57-61). Domains I and II are largely asialylated andmono-sialylated structures, while Domains III and IV and V are largelydi-and tri-and tetra-sialylated structures.

It was empirically observed that the percentage of the total profile inDomains III and IV correlated well with the AUC for all of the samples,including the Process X material (FIG. 51). Most of the structures inthese domains are expected to be fully sialylated and galactosylated.Using data from the M-1 group, a Domain III and IV percentage of about29% should have the same clearance rate as the Process X material.Domain III and IV data from Method 2 is typically about 4% lower (21%vs. 25%) than data generated by Method 1.

In addition, the percentage of the total profile in Domains I and IIalso were observed to correlate well with the AUC for all of the samples(FIG. 56). Most of the structures in these domains are expected to belargely asialylated and mono-sialylated structures. FIG. 56 shows thatthe clearance rate was higher in samples with a lower percentage ofDomains I and II versus those samples with a higher percentage ofDomains I and II. The decreased glycosylation of Domains I and IIcorrelate with the presumably increased presence of peptidesglycosylated in Domains III and IV.

Although the sialic acid to protein molar ratio has been traditionallyused to predict the clearance rate for CTLA4Ig, different fermentationmedia have produced molecules with the same NANA ratio but differentclearance rates in monkey PK studies. To develop a better way to predictclearance rates, two other sets of analytical data (monosaccharideanalysis and carbohydrate profile) were evaluated and compared to monkeyPK data. The most predictive analytical parameter was the extent ofgalactosylation of CTLA4Ig. To reduce analytical variability of thisassay, the galactose molar ratio was normalized to the mannose molarratio. The resulting Gal:Man ratio correlated well with the AUC resultsfrom the monkey PK study for all of the samples, including the Process Xmaterial. This result is consistent with a model that the clearance rateof CTLA4Ig is primarily determined by the number of exposed terminalGlcNAc residues on the molecule. If this model is correct, the extent ofgalactosylation should predict clearance rates better than sialylation.To ensure pharmacokinetic comparability (AUC >75% of material from the XProcess), a specification for the galactose to mannose ratio ofGal:Man >0.65 is recommended for purified bulk CTLA4-Ig drug substance(BDS).

When only material prepared by the Y Process or CD-CHO1 process isanalyzed, the NANA to protein molar ratio can predict clearance ratesaccurately. This relationship does not apply, however, to materialprepared by the X Process. To be comparable to X Process material,CTLA4-Ig prepared by the Y Process or the CD-CHO1 process must have aNANA ratio 2 units higher (NANA=9 for CD-CHO1 is comparable to NANA=7for the X Process material). Because the sialic acid assay is accurateand has a rapid turn-around time, it is useful as an in-processanalytical tool to monitor the quality of the CTLA4Ig during afermentation run.

To maintain comparable pharmacokinetics (AUC >75% of reference), aspecification of NANA >8 is recommended for purified BDS. This valuealso represents a reasonable target for harvesting the fermentationruns. Because the purification process can increase the sialic acidratio by at least 2 units, setting the harvest target at NANA=8 willensure an actual harvest value of at least NANA=7. The purificationprocess can increase this value to at least NANA=9, which is comparableto the Process X material and well above the aforementioned recommendedminimum specification of the BDS.

The carbohydrate profile also predicted the monkey PK results well forboth Process X material and Process Y material when the percent areaunder Domains III and IV was compared to the AUC. Domains III and IVconsist largely of fully sialylated and galactosylated carbohydratestructures.

The data in Table 56 can be further presented so as to show acorrelation between the AUC value, the NANA value, the Gal value, andthe total percentage of sum of the AUC of Domains II and IV. See below:

Sum of Domains III AUC NANA Gal and IV 2,337 2.3 4.1 9.7 8,832 7.1 8.119.7 7,266 7.3 11.1 22.0 9,425 7.9 11.5 22.0 7,765 6.9 9.1 23.4 15,7798.8 11.4 26.8 18,750 10 13.0 28.2 17,060 6.9 13.1 28.5 15,753 15,45917,739 10.3 12.6 29.7 20,445 9.9 15.8 33.4 20,707 9.8 12.8 36.2

[001357] This table above shows that there is a correlation between thesum of Domains III (and IV) and the PK outcome of the composition. TheAUC of Domains III and IV and V are directly related to the molar ratioof NANA and Gal to moles of CTLA4-Ig protein. Therefore, the inventionprovides for compositions characterized in that their carbohydrateprofile contains a sum of Domains III and IV, or a sum of Domains III,IV and V of from 18 to about 37 AUC %. In one embodiment, the sum ofDomains II, IV and V is about 19 to about 36, is about 20 to about 35,is about 21 to about 34, is about 22 to about 33, is about 23 to about32, is about 24 to about 31, is about 25 to about 30, is about 26 toabout 29, is about 27 to about 28 AUC %. In one embodiment, theinvention provides for CTLA4-Ig compositions characterized in that theDomain III has an AUC % of the total of 19±4; and Domain IV has an AUC %of the total of 7±4.

Example 43 Tryptic Peptide Mapping of CTLA4-IG

CTLA4-Ig derived from transfected Chinese Hamster Ovary (CHO) cells is aglycoprotein with a molecular mass of approximately 92500 Daltons.Peptide mapping is a highly sensitive method for determining theidentity of the primary structure of a protein and is useful indetecting post-translational modifications. The protein is denaturedusing guanidine-HCl, reduced, and alkylated using DTT and IAA. Theprotein is desalted using NAP-5 columns and the digest mixture isanalyzed by reversed phase (C18) chromatography. Peak detection is doneby UV absorbance at 215 nm.

REAGENTS: Mobile Phase A solution (0.02% Trifluoroacetic Acid (TFA) inWater (v/v)); Mobile Phase B solution (0.02% TFA in 95% ACN(Acetonitrile) and 5% Water (v/v)); Alkylating Agent (200 mMIodoacetamide (IAA)); Dilution Buffer (100 mM Tris, 25 mM NaCl, pH 8.0);Denaturing Buffer (8 M Guanidine, 50 mM TRIS, pH 8.0); Digestion Buffer(50 mM TRIS, 10 mM CaC1₂, pH 8.0); Reducing Agent (100 mM DTT).

INSTRUMENTATION: (equivalent instrumentation may be used) NAP-5 columns(Amersham, cat. # 17-0853-02); HPLC Column Heater; Water's Alliance HPLCsystem with column heater and UV detector.

Reduction and Alkylation: Samples (for example, CTLA4^(A29YL104E)-Ig,standards, etc.) were diluted to 10 mg/ml by adding water to a finalvolume of 100 μL (1 mg). 560 μl of denaturing buffer and 35 μL ofReducing Agent (100 mM DTT) were added to the 100 μl samples, weremixed, and spun down in a microcentrifuge for 3 seconds. Samples werethen incubated at 50° C. for 20 minutes±2 minutes. 35 μL of AlkylatingAgent (200 mM IAA) was then added to each sample, and again samples weremixed, and spun down in a microcentrifuge for 3 seconds. Samples weresubsequently incubated at 50° C. for 20 min.±2 minutes, in the dark.After the NAP-5 columns were equilibrated by pouring 3 columns volumes(about 7-8 mL) of digestion buffer, 500 μl of the reduced and alkylatedmixtures were poured over the NAP-5 columns, allowing the liquid todrain through column. Samples were then collected from the NAP-5 columnsvia eluting sample off of the column with 1 mL of digestion buffer.

Digestion: Samples were digested with 20 μL of trypsin (0.5 μg/μL) in38° C. water bath for 4 hours (±0.5 hr). Upon completion of digest,samples were acidified with 2.5 μL of TFA. Samples were then placed intoautosampler vials for subsequent analysis.

Instrument Method: The instrument method is shown below:

Time (min) Flow (mL/min) Mobile Phase A Mobile Phase B 0 0.7 100 0 170.7 83 17 27 0.7 78 22 42 0.7 73 27 58 0.7 65 35 74 0.7 52 48 79 0.7 0100 84 0.7 100 0 88 0.7 100 0

The column was equilibrated with 100% Mobile Phase A buffer for 25minutes prior to the first injection. UV absorbance was monitored at 215nm while column temperature was manintained at 37° C. and theautosampler temperature at 4° C. A mobile phase A buffer blank was runbefore the first system suitability standard, thereafter followed by asingle 50 μL injection of each sample. A reference material injectionshould bracket every six-sample injections. The peptide map chromatogramgenerated for a CTLA4-Ig sample is depicted by FIG. 52. The retentiontime differences for peaks T2, T3, T15, and T19 (FIG. 52 and Table 57)between the initial and bracketing reference material chromatograms mustbe±0.5 min.

TABLE 57 Theoretically Expected Fragments of CTLA4-Ig Digested withTrypsin Fragment Monoisotopic Average No. Residue No. Mass Mass SequenceT1  1-14 1464.8 1465.7 MHVAQPAVVLASSR T2 15-28 1484.7 1485.6GIASFVCEYASPGK T3 29-33 574.3 574.6 ATEVR T4 34-38 586.4 586.7 VTVLR T5*39-83 4895.2 4898.3 QADSQVTEVCAATYMMGNELTFLDDSICTGT SSGNQVNLTIQGLR T684-93 1170.5 1171.4 AMDTGLYICK T7*  94-128 3993.9 3996.4VELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQ EPK T8** 129-132 435.2 435.4 SSDK T9**133-158 2687.4 2689.1 THTSPPSPAPELLGGSSVFLFPPKPK T10 159-165 834.4 835.0DTLMISR T11 166-184 2138.0 2139.3 TPEVTCVVVDVSHEDPEVK T12 185-198 1676.81677.8 FNWYVDGVEVHNAK T13 199-202 500.3 500.6 TKPR T14* 203-211 1188.51189.2 EEQYNSTYR T15 212-227 1807.0 1808.1 VVSVLTVLHQDWLNGK T16 228-230438.2 438.5 EYK T17 231-232 306.1 306.3 CK T18 233-236 446.2 446.5 VSNKT19 237-244 837.5 838.0 ALPAPIEK T20 245-248 447.3 447.5 TISK T21249-250 217.1 217.3 AK T22 251-254 456.2 456.5 GQPR T23 255-265 1285.71286.5 EPQVYTLPPSR T24 266-270 604.3 604.7 DELTK T25 271-280 1160.61161.4 NQVSLTCLVK T26 281-302 2543.1 2544.7 GFYPSDIAVEWESNGQPENNYK T27303-319 1872.9 1874.1 TTPPVLDSDGSFFLYSK T28 320-324 574.3 574.7 LTVDKT29 325-326 261.1 261.3 SR T30 327-349 2800.3 2802.1WQQGNVFSCSVMHEALHNHYTQK T31 350-356 1659.3 659.7 SLSLSPG *ContainsN-linked carbohydrate **Contains O-linked carbohydrate

Number of Theoretical Plates: Column efficiency, evaluated as the numberof theoretical plates, can be measured quantitatively using theretention time and the width of peak according to the Equation:

$N = {16\left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   “w” is the peak width at the baseline measured by extrapolating        the relatively straight sides to the baseline, “t” is the        retention time of the peak measured from time of injection to        time of elution of peak maximum.

If the N <50000, re-equilibrate the column.

Resolution: The resolution (R) between 2 peaks, for example peak T30 andpeak T12 as indicated in FIG. 52, can be determined using the followingequation:

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{\left( {w_{1} + w_{2}} \right)}$

-   -   Where:    -   t₁, t₂=retention times of fragments peak T30 and peak T12,        respectively    -   w_(l), w₂=tangent-defined peak width at baseline of the peaks        with retention times t₁ and t₂, respectively.

If R <1.5, the column should be re-equilibrate and if the problempersists, the column should be replaced.

Exemplary values: The difference between the relative peak areas forpeaks T3, T15, and T19 in the test article and reference material mustbe ≤10.0%. The relative peak area of a peak is defined as the peak areaexpressed as a percentage of the peak area of peak T2. The differencebetween the relative peak areas of the test article and the initialsystem suitability reference is obtained as shown below. The relativepeak area (R_(SX)) can be calculated for each of the peaks T3, T15, andT19 in the chromatogram of the test article by using the formula:

R_(SX)=(A_(SX)/A_(S2))*100

-   -   Where:    -   R_(SX)=relative peak area of peak X in the chromatogram    -   A_(SX)=area of peak X in the sample and    -   A_(S2)=area of peak T2 in the sample.

Similarly, the relative peak areas (R_(RX)) can be calculated for eachof the peaks T3, T15, and T19 in the chromatogram of the standard. Thedifference between the relative peak areas in the sample and thestandard (DX) can subsequently be calculated by using the formula:

D _(X)=[(R _(SX) −R _(RX))/(R _(RX))]*100

If a single additional peak is present in the sample, the relative peakheight for that peak as compared to peak T11 can be determined by usingthe following formula:

Relative peak height R_(T)=(H_(T)/H₁₁)*100 where

-   -   H_(T)=height of the peak with retention time t min    -   H₁₁=height of peak T₁₁, the tallest peak in the chromatogram.

In one embodiment, if the relative peak height of the new peak is 5.0%,then the profile is considered to be consistent with the profile ofCTLA4-Ig standard. If the relative peak height of the new peak is >5.0%,then the profile is considered to be not consistent with the profile ofthe CTAL4-Ig standard material.

The percent oxidation data was acquired by use of a RP-HPLC trypticmapping assay that quantifies the area percent oxidation of Met85 in theprotein to methionine sulfoxide. Percent oxidation in the method isobtained by measuring UV peak areas in the RP-HPLC tryptic map for theT6 tryptic peptide, comprised of residues 84-93 containing Met85, andthe corresponding oxidized tryptic peptide, T6ox, containing Met(O)85.The area percent oxidation of Met85 to Met(O)85 is proportional to thearea percent of the T6ox peak:

Percent Oxidation=100*A_(T6ox)/(A_(T6ox)+A_(T6))

where,

-   -   A_(T6)=peak area for T6 tryptic peptide, (84-93).    -   A_(T6ox)=peak area for T6ox tryptic peptide, Met(O)⁸⁵(84-93).

The percent deamidation data was acquired by use of a RP-HPLC trypticmapping assay that quantifies the area percent oxidation of deamidationin the assay is obtained by measuring UV peak areas in the RP-HPLCtryptic map for the T26 tryptic peptide, comprised of residues 281-302containing Asn294, and the corresponding deamidated tryptic peptide,T26deam1, containing isoAsp294. The area percent deamidation of Asn294to isoAsp294, then, is proportional to the area percent of the T26deam1peak:

${{Percent}\mspace{14mu} {Deamidation}} = {100*\frac{A_{T\; 26\; {deam}\; 1}}{\begin{matrix}{A_{{T\; 26}\;} + A_{T\; 2\; 6\; {deam}\; 1} + A_{T\; 26\; {deam}\; 2} +} \\{A_{T\; 26\; {deam}\; 3} + A_{T\; 26\; {deam}\; 4}}\end{matrix}}}$

where,

-   -   A_(T26)=peak area for T26, (281-302)    -   A_(T26deam1)=peak area for T26deam1, isoAsp²⁹⁴(281-302).    -   A_(T26deam2)=peak area for T26deam1, Asp299(281-302).    -   A_(T26deam3)=peak area for T26deam3, Asp²⁹⁴(281-302).    -   A_(T26deam4)=peak area for T26deam4, Asu²⁹⁴(281-302).

Example 44 CTLA4-I2 N-Linked Oligosaccharide Carbohydrate Profiling byHigh Performance Anion Exchange Chromatography with ElectrochemicalDetection

The carbohydrate structures present on glycoproteins can affect theirfunction and in vivo clearance. It is therefore important to monitor thestructural consistency of the carbohydrates of recombinantly producedbatches of glycoproteins. CTLA4-Ig is a recombinant protein containingboth N-linked and O-linked (serine-linked) glycosylation sites. Here,N-linked (asparagine-linked) carbohydrates present on CTLA4-Ig aremonitored. In this method, oligosaccharides are cleaved by enzymaticdigestion with PNGase F (Peptide: N-Glycosidase F), then isolated byreversed-phase HPLC in a two-column system, separated by highperformance anion exchange chromatography (HPAEC), and monitored byelectrochemical detection (integrated amperometry). The chromatogramgenerated is the N-linked carbohydrate profile, wherein profiles ofCTLA4-Ig samples should be similar to such.

This method describes the procedure to determine the HPAEColigosaccharide profile of N-linked oligosaccharides released fromCTLA4-Ig samples. A purpose of the method is to provide chromatographicprofiles of CTLA4-Ig drug substance N-linked oligosaccharides which canbe used for comparative analysis between. The glycosylation on theCTLA4-Ig contains N-linked oligosaccharides. These oligosaccharides areliberated by enzymatic hydrolysis with PNGase F over the course of 22hours. The free oligosaccharides are profiled using high pH anionexchange chromatography employing electrochemical detection.Oligosaccharide profiles of drug substance are evaluated againstconcurrently run samples of reference material. Results are reported aspercent deviation of selected domains and peaks from the same peaks inthe reference standards.

% Diff Percent Difference % RSD Percent Relative Deviation HPAEC High pHAnion Exchange Chromatography HPLC High Performance LiquidChromatography NaOAc Sodioum Acetate NaOH Sodium Hydroxide PNGase FPeptide: N-Glycosidase F

MATERIALS. Equivalent materials may be substituted unless otherwisespecified.

Waters Total Recovery Vials with bonded Waters Corporation, CatalogPTFE/silicone septa No. 186000234 Microcon YM 10 Centrifugal Millipore,Catalog No. 42407 Filter Devices RapiGest SF Waters Corporation, CatalogNo. 186001861

INSTRUMENTATION AND CONDITIONS. Equivalent instrumentation may be usedunless otherwise specified.

Instrumentation:

Alliance HPLC system Waters Corporation equipped with: Autosampler(refrigerated), Eluent Degas Module Model 2465 Electrochemical DetectorColumn: CarboPac PA-1 4 × 250 mm Dionex Corporation, Catalog No. 35391Guard Column: CarboPac PA-1 4 × Dionex Corporation, Catalog 50 mm No.43096 Empower Data Collection system Version 3.2 or current validatedBMS version

Chromatography Conditions for Oligosaccharide Profile by Anion-ExchangeChromatography

Column Temperature 29° C. Flow Rate 1 mL/min Mobile Phases and GradientGradient Program Conditions Time (min) %1 %2 %3 1: 500 mM NaOAc Initial0 30 70 2: 400 mM NaOH 0.0 0 30 70 3: HPLC Grade Water 11.0 0 30 70 12.04 30 66 20.0 10 30 60 80.0 45 30 25 81.0 0 30 70 100 0 30 70 Waters 2465settings Mode Pulse Empower settings Range = 5 μA E1 = +0.05 V E2 =+0.75 V E3 = −0.15 V t1 = 400 msec t2 = 200 msec t3 = 400 msec Samplingtime(ts) = 100 msec Time constant(filter)t = 0.1 sec Range offset = 5%Polarity + Temperature = 29° C. NOTE: Equilibrate the column anddetector with the initial mobile phase at the analysis flow rate forapproximately 2 hours, or until baseline is stable before makinginjections.

Autosampler Temperature 4° C. set to: Injection Volume 60 μL Run Time100 minutes Approximate Retention Times (RT; minutes) of dominant peaksin each Domain (see FIG. 1); values may vary depending on RT of SystemSuitability (SS) Standard Approximate RTs (min) SS: 18.5 Peak 1A: 20.0Peak 1B: 20.8 Peak 1C: 21.4 Peak 1D: 22.4 Peak 1E: 23.1 Peak 2 31.5 Peak3: 44.8 Peak 4: 58.5

Electrode Cleaning (Waters 2465). Follow cleaning instructions in thedetector manual. Use the diamond slurry provided in the flow cell kit topolish the surface of the electrode(s). If polishing does not yieldacceptable results, replace the electrodes with a new flow cell kit.Re-build the flow cell using a new spacer (50 μm).

REAGENTS. NOTE: Label and document all reagent preparations according todepartmental procedures.

Preparation of Mobile Phases for HPAEC Oligosaccharide CarbohydrateProfiling.

HPAEC Eluent 1: 500 mM Sodium Acetate (NaOAc). Weigh 20.51±0.05 g ofSodium Acetate (anhydrous) into a 500 mL graduated cylinder containing400 mL of HPLC grade water. Bring volume to 500 mL with HPLC grade waterand stir for 5 minutes using a plastic serological pipette untilcompletely mixed. Filter the solution through a 0.2 μm nylon filter.Transfer to a 1 L eluent bottle. Cap the bottle loosely and sparge withhelium for 20 minutes. Tighten cap and pressurize the bottle withhelium. Store solution at room temperature under helium for up to threeweeks.

HPAEC Eluent 2: 400 mM Sodium Hydroxide (NaOH). Using a 1 L graduatedcylinder, measure 960 mL of HPLC grade water and transfer to a clean 1 Leluent bottle. Using a serological plastic pipet, add 40.0 mL of 10 NNaOH directly into the eluent bottle and mix the eluent by swirling. Capthe bottle loosely and sparge with helium for 20 minutes. Tighten capand pressurize the bottle with helium. Store solution at roomtemperature under helium for up to three weeks.

HPAEC Eluent 3: HPLC grade Water. Fill a 1 L eluent bottle withapproximately 1 L of HPLC grade water. Place eluent bottle on system,cap loosely, and sparge for approximately 20 minutes. Tighten cap andpressurize the bottle with helium. Store solution at room temperatureunder helium for up to three weeks.

50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH=7.5. Sodium Azide(NaN₃) should be handled with care to avoid inhalation (toxic) andcontact with skin (irritant). Consult the MSDS sheet for additionalrequirements. After weighing of NaN₃, the balance area should bethoroughly cleaned.

NaH₂PO₄•H₂O 6.9 g Na N₃ 0.2 g H₂O 1.0 liter final volume

Weigh out 6.9 g±0.1 g of NaH₂PO₄.H₂O and 0.2 g NaN₃ and dissolve in 800mL of HPLC grade H₂O in a 1 L reagent bottle using continuous mixingwith a magnetic stirring bar. Using a pH meter, adjust the pH of thesolution to 7.5 using 10M NaOH. Bring the final volume to 1.0 literusing a 1 L graduated cylinder. Store solution at room temperature forup to six months. PNGase F Enzyme Working Stock in 50 mM SodiumPhosphate Buffer, 0.02% Sodium Azide, pH=7.5.

50 mM Sodium Phosphate Buffer

0.02% Sodium Azide, pH = 7.5. 1.8 mL PNGase F from Kit, Catalog No.P0704L 0.2 mL

Pipette 1.8 mL of 50 mM Sodium Phosphate Buffer, 0.02% Sodium Azide, pH7.5 into a 1.8 mL cryogenic vial. Add 0.2 mL of PNGase F from kit andmix thoroughly. Store solution at −20° C. or less for up to six months.The solution may be aliquoted prior to freezing.

External System Suitability Standard. Stachyose Stock Solution (1.25mg/mL): Weigh 0.125 g of Stachyose onto a weighing paper. Using ananalytical balance and transfer to a 100 mL volumetric flask. Fill tomark with HPLC grade water and mix thoroughly. Aliquot in 2 mL portionsinto Nalgene cryovials. Store solution at −20° C. or less for up to sixmonths.

Stachyose System Suitability Standard (12.5 μg/mL): Pipet 1 mL of the1.25 mg/mL stock into a 100 mL volumetric flask. Fill to mark with HPLCgrade water and mix thoroughly. Aliquot in 200 μL portions into 0.65 mLmicrofuge tubes. Place tubes in appropriately labeled storage box. Storesystem suitability solution at −20° C. or less for up to six months.

Standard and Sample Preparation

Reference Material Preparation. To a vial containing 1 mg of lyophilizedRapiGest SF, add 625 μL of 50 mM NaPhosphate buffer containing 0.02%NaAzide, pH 7.5. NOTE: A single pool of RapiGest SF containing buffershould be used for all samples within a sample set. Several vials ofRapiGest SF may be reconstituted and combined to provide adequatevolume. To a 0.65 mL Eppendorf tube add 120 μL of the RapiGest SFcontaining buffer. Add 40 μL of Reference Material (˜50 mg/mL). Thefinal RapiGest SF concentration should be 0.12% w/v. Add 40 μL of thePNGase F working stock, mix thoroughly, spin down the sample, and placeat 38±2° C. for 22±2 hours (water bath or the Alliance autosamplercompartment). Pipet sample into a microcon YM-10 centrifugal filter andcentrifuge at 13,000 g for 30 minutes. Place 200 μL of HPLC water in thefilter and rinse into the filtrate by centrifuging for an additional 30minutes at 13,000 g. Vortex the combined filtrate for 15 seconds andcentrifuge the sample for 10 seconds. Using a pipette transfer theresulting solution (˜380 μL) to an HPLC total recovery autosampler vial.

Sample Preparation: To a 0.65 mL Eppendorf tube add 120 μL of theRapiGest SF containing buffer. Add 40 μL of protein sample (this volumeshould equate to between 1 and 2 mg of CTLA4-Ig). The final RapiGest SFconcentration should be 0.12% w/v. Add 40 μL of the PNGase F workingstock mix thoroughly by vortexing for 10 seconds. Spin down the sample,and place at 38±2° C. for 22±2 hours (water bath or the Allianceautosampler compartment). Pipet sample into a microcon YM-10 centrifugalfilter and centrifuge at 13,000 g for 30 minutes. Place 200 μL of HPLCwater in the filter and rinse into the filtrate by centrifuging for anadditional 30 minutes at 13,000 g. Vortex the combined filtrates for 15seconds and centrifuge the sample for 10 seconds. Transfer the resultingsolution (˜380 uL) to a total recovery HPLC autosampler vial.

Electrochemical Detector Cell Stabilization: Inject 30 μL of theexternal stachyose system suitability standard (12.5 μg/mL). Ensure thepeak height for stachyose is ≥800 nA. Ensure there is no excessiveelectrical noise from the cell and the baseline is flat. If thestachyose sensitivity or the baseline is unacceptable, check the buffercomposition, clean the electrode or replace the electrode. If excessivenoise is present, check cell to ensure removal of all air bubbles.Restabilize the cell and re-inject stachyose standard. If problemspersist, take other appropriate actions or contact your supervisor.

Theoretical Plates (N): Determine the number of Theoretical Plates (N)based on the Stachyose peak using the formula below. This is donethrough the Empower data analysis system or may also be done manually.

N=16(t/W)²

WHERE:

-   -   t: retention time measured from time of injection to peak        elution time at maximum height    -   W: width of peak by extrapolation of sides to baseline.    -   N must be 6000. If the plate count is less than 6000, adjust the        run gradient or replace column.

Tailing Factor (T): Determine column Tailing Factor (T) based on theStachyose peak using the formula below. This is done through the EMPOWERdata analysis system or may also be done manually.

T=9W_(0.05)/2f)

WHERE:

-   -   W₀₀₅: width of peak at 5% of height (0.05 h).    -   f: the measurement (width) from front edge of peak at W₀₀₅ to        the apex of the peak.

T must be ≤1.2. If the tailing factor is greater than 1.2, check buffercomposition, replace the column or clean the column and re-inject systemsuitability standard.

Stachyose System Suitability Standard Retention Time Verification: Theretention time is system dependent. The stachyose system suitabilitystandard should exhibit a retention time of 18.5±2.0 minutes.

CTLA4-Ig Reference Material-Observe the carbohydrate profile from thefirst bracketing reference material injected prior to injection ofsamples. The carbohydrate profile should be similar to that shown inFIG. 67. Absolute retention times are system dependent. Ensure that thedifference in retention times between the first peak in Domain I (Peak1A) and the main peak in Domain III (Peak 3) is between 22 minutes and28 minutes. If delineation of peaks does not resemble that obtained inFIG. 67 take appropriate actions (e.g. check instrument function, cleancolumn, check/replace buffers, replace column) and re-evaluate. Thefollowing procedure may be used to clean the column: turn off the celland clean the column with 80% Eluent 1, 20% Eluent 2 for 5 minutesfollowed by 50% Eluent 1, 50% Eluent 2 for 10 minutes. Re-equilibratethe column and cell (with cell turned on) at initial conditions andre-evaluate.

Injection Sequence:

Set up the injection sequence of isolated oligosaccharides as follows:

-   -   Stachyose Standard (30μL)    -   Reference Material (60 μL)    -   Sample(s) (60 μL)    -   Reference Material (60 μL)

It is recommended that ≤five samples be run between bracketing referencematerial injections.

DATA ANALYSIS: Process the Chromatograms. Process the chromatograms forthe Reference Material and samples in EMPOWER. Set integrationparameters so that peak delineation and the baseline is similar to thatshown in FIG. 67, integration lines may need to be placed manually.Perform calculations for relative Domain areas and relative peak areas(see tables included in Brief Description of FIG. 67 and at the end ofthis example). Determine the average values for these parameters for theReference Material and for each sample if replicate injections weremade.

For the Reference Material, determine relative deviation for Domains I,II, III, Peaks 1A and 1B for each replicate with respect to the averageof all replicates.

Comparison of Profiles of Sample to Reference Material Profiles.

Visual Comparison Determine if both samples and Reference Material havethe same number of Domains and primary peaks. Primary peaks are thosepeaks labeled in the description of FIG. 67 (Peaks 1A, 1B, 1C, 1D, 2, 3and 4). Relative Quantitation Comparison. Compare the relative areas ofsamples (Domains I, II, and III and Peaks 1A, and 1B; if replicateinjections were made of samples use their average values) with theaverage relative areas from the bracketing Reference Materialinjections. Determine the relative difference of these areas from theaverage Reference Material values.

Calculations. % Domain Area (Relative Domain Area): Calculate the %Domain area for the Domains of the profiles for the Reference Materialand samples. Refer to FIG. 67 for pattern of Domain areas. Following theexample in FIG. 67, calculate the Domain percent ratios by using thefollowing information and formula (retention times are system dependentand reflect result in FIG. 67:

-   -   Domain I: Sum of the peak areas at approximate retention times        18-24 minutes (Peaks 1A-1E)    -   Domain II: Sum of the peaks from 26-38 minutes    -   Domain III: Sum of the peaks from 39-50 minutes    -   Domain IV: Sum of the peaks from 51-64 minutes    -   Domain V Sum of the peaks from 65-75 minutes

NOTE: Retention time windows for Domains will shift according tovariations in daily chromatographic performance. Adjust timesaccordingly.

${{Domain}\mspace{14mu} {Area}\mspace{14mu} \%} = {\frac{{Individual}\mspace{14mu} {Domain}\mspace{14mu} {Area}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {Domain}\mspace{14mu} {Area}} \times 100\%}$

-   -   For Domains I-III also calculate the average values in the        bracketing reference material injections, as well as in samples        if replicate injections are made.

% Peak Area (Relative Peak Area). Calculate the % peak area for Peaks1A, 1B, 1C, and 3 of the profiles for the Reference Material andsamples. Refer to FIG. 67 for pattern of peak areas; retention times aresystem dependent. Calculate the peak percent ratios by using thefollowing information and formula:

${{Individual}\mspace{14mu} {Peak}\mspace{14mu} {Area}\mspace{14mu} \%} = {\frac{{Individual}\mspace{14mu} {PeakArea}}{{Sum}\mspace{14mu} {of}\mspace{14mu} {all}\mspace{14mu} {Domain}\mspace{14mu} {Area}} \times 100\%}$

For each of Peaks 1A and 1B, also calculate the average values in thebracketing reference material injections, as well as in samples ifreplicate injections are made.

Calculation of the Percent Difference from Average Reference MaterialValues. Use the following formula to calculate percent differences inaverage relative areas of Domains I-III, Peaks 1A and 1B of samplescompared to Reference Material:

% Diff=|RM−S|/RM×100

-   -   WHERE:    -   RM=average relative area value of interest for Reference        Material    -   S=average relative area value of interest for a sample    -   | | =absolute value

Results: Results are to be reported are the calculated percentdifference from reference material for Domain I, Domain II, Domain II,peak 1A and peak 1B. Include an integrated representative chromatogramfor both the Reference Material and the sample. Include the relativearea percentages of Domains I-III and Peaks 1A and 1B to a tenth of apercent for both sample and Reference Material (average of bracketinginjections). Additionally, for each of the bracketing Reference Materialinjections, the % Domain Areas for Domain I, II and III and % Peak Areasfor Peak 1A and 1B should be within 15% of their average values.

FIGS. 56-61, 67, 75 and 81-83 show resultant data from N-linkedoligosaccharide profiles as are described herein. FIG. 67 depicts atypical N-Linked Oligosaccharide Profile (Domains I, II, III, IV and V,and Peaks 1A and 1B within 5% of Lot averages). Peaks 1A, 1B and 1Crepresent the asialo N-linked oligosaccharide structures of G0, G1 andG2.

Peak Height Area Retention Area Relative Percentage Domain/ TimePercent- to Tallest of Parent Domain Peak (minutes) age Peak DomainComposition Domain I 19.413 31.3 5 Peaks Domain II 29.076 33.2 5 PeaksDomain III 42.819 24 5 Peaks Domain IV 55.899 9.4 6 Peaks Domain V67.546 2.2 6 Peaks Peak 1A 19.413 7.3 89.8 23.3 Peak 1B 20.29 10.7 10034.2 Peak 1C 21.032 8.8 94.3 28.1 Peak 1D 21.925 2.8 27.5 8.95 Peak 1E22.685 1.7 11.8 5.43 Peak 2 30.763 18.3 88.9 55.1 Peak 3 43.823 14.557.8 60.4 Peak 4 57.368 4.4 20.1 46.8

FIG. 67 shows a typical N-linked carbohydrate profile for a CTLA4-Igcomposition. The table directly above shows tabulated data for theN-linked oligosaccharide profile of CTLA4-Ig.

The table directly below shows observed ranges of CTLA4-Ig.

Minimum Area Maximum Area Domain/Peak % % Domain I 24.5 35.2 Domain II26.3 34.1 Domain III 21.9 31.5 Domain IV + V 7.9 18.6 Peak 1A 4.5 11.2Peak 1B 8.7 11.8

Example 45 An IN-VITRO Cell based Bioassay for CTLA4-Ig

T cells require two signals for activation and subsequent proliferation.The first signal is provided by the interaction of an antigenic peptidewith the TCR-CD3 complex. The second co-stimulatory signal occurs withthe interaction between CD28 on the T cell and the B7 protein on anantigen-presenting cell. Upon receipt of these two signals, T cellssecrete the cytokine Interleukin 2 (IL-2). Release of IL-2 leads tocellular activation and proliferation. CTLA4-Ig, a soluble,immunosuppressive compound, also binds to the B7 protein on the antigenpresenting cell, thus blocking functional interaction with CD28 andpreventing the co-stimulatory signal that is necessary for IL-2production.

In this method, Jurkat T cells transfected with the luciferase gene,under the control of the IL-2 promoter, are co-stimulated with Daudi Bcells in the presence of anti-CD3. The co-stimulation activates the IL-2promoter, which in turn produces luciferase protein. The resultingluminescent signal is measured using a Luciferase Assay System. In thissystem, CTLA4-Ig produces a dose-dependent decrease in luciferaseactivity.

This method examines the effect of CTLA4-Ig on the co-stimulatory signalneeded for IL-2 production. The presence of soluble CTLA4-Ig preventssignaling between the T cell and antigen-presenting cell. Without thissignal, IL-2 is not produced, thus preventing the clonal expansion of Tcells. A vector with the luciferase gene was created using the IL-2promoter. Jurkat T cells were then transfected with this reportervector. A positive clone, Jurkat.CA, was selected and used in themethod.

This bioassay involves stimulating transfected T cells (Jurkat.CA) withanti-CD3 and B cells (Daudi). Co-stimulation provided by the B cells isinhibited by the addition of CTLA4-Ig. Jurkat.CA and Daudi cells areseeded into the wells of a 96 well, white, opaque, flat-bottom plate andstimulated with anti-CD3 in the presence of different concentrations ofCTLA4-Ig. After a 16 to 20 hour incubation at 37° C., the wells areassayed for luciferase activity. Inhibition of co-stimulation byCTLA4-Ig is seen as a dose-dependent decrease in luciferase activity.FIG. 95 shows a procedure flow chart.

REAGENTS: Daudi Cell Culture Media (10% fetal bovine serum, 1% MEMsodium pyruvate in RPMI 1640); Jurkat.CA Cell Culture Media (10% calfserum, 1% MEM sodium pyruvate, 400 μg/mL of geneticin in RPMI 1640);Bioassay Media (0.2 μg/mL of anti-CD3 antibody and 1%penicillin-streptomycin solution in Daudi Cell Culture Media);Bright-Glo Luciferase Solution from assay system (Promega, Catalog #E2620).

INSTRUMENTATION: Nikon, Diaphot 200 Inverted microscope; PackardTopCount NXT Luminometer; Tecan Genesis Liquid Handler; Coulter Vi-CellCell Counter; Zymark RapidPlate-96.

Preparation of Working Solutions: 3 mL of CTLA4-Ig solutions (5000ng/mL) in bioassay media.

Eight point curves were prepared for the standard, quality control, andsamples at the concentrations of 100, 4, 2, 1, 0.5, 0.2, 0.1, and 0.002μg/mL CTLA4-Ig as shown in Table 58 below for final concentrations inthe assay, after two-fold dilution into the plate, of 50, 2, 1, 0.5,0.25, 0.1, 0.05, and 0.001 μg/mL.

TABLE 58 Dilutions used to generate standard curves. Standard QualityCurve Point Curve Control Sample 1 Sample 2 1 100 μg/mL 100 μg/mL 100μg/mL 100 μg/mL 2 4 4 4 4 3 2 2 2 2 4 1 1 1 1 5 0.5 0.5 0.5 0.5 6 0.20.2 0.2 0.2 7 0.1 0.1 0.1 0.1 8 0.002 0.002 0.002 0.002

200,000 cells were added per well of a 96 well plate and were incubatedat 37° C., 5% CO₂, and 85% humidity. 12×10⁶ Jurkat.CA cells and 12×10⁶Daudi cells were combined in a sterile centrifuge tube. The cells werecentrifuged at ˜125×g for 10 minutes at room temperature and werethoroughly re-suspend in 9 mL of Daudi cell culture media by gentlypipetting repeatedly with a serological pipet until no cell clumps werevisible to give a concentration of 2.7×10⁶ cells/mL. 75 μL of eachsolution from Table 58 was added to the appropriate wells of the platecontaining cells. The plate(s) were then sealed with TopSeal-A andincubated at 37° C., 5% CO₂, and 85% humidity for 16 to 20 hours. Afterthe plates and Bright-Glo luciferase solution were equilibrated to theinstrument temperature, 150 μL of Bright-Glo luciferase solution wasadded to each well simultaneously and were mixed. A plate is then placedin the TopCount NXT immediately after mixing for equilibration in thedark for 10 minutes. The luminescent signal was then measured in aTopCount NXT using a 1 second integration per well or as appropriate tothe particular type of luminometer used.

The output from the TopCount NXT was recorded, read into a standardanlysis program, and data were transformed by taking their logarithm(base 10). The transformed data from each article were fit to afour-parameter logistic model as shown in the equation below:

$\begin{matrix}{{{Log}_{10}\left( y_{jk} \right)} = {D + \frac{\left( {A - D} \right)}{1 + \left( \frac{x_{j}}{C} \right)^{B}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

-   -   Where:    -   A is the top plateau of the curve, D is the bottom plateau of        the curve, B is the slope factor, and C is the concentration        that produces an effect equal to the average of A and D.

An R² statistic, and a lack-of-fit F-test can be calculated for eacharticle. A ratio of the minimum, maximum and slope of the test articlesrelative to the standard material can also be calculated. In addition,confidence intervals for the ratios can also be computed.

The relative potency of each article was determined by fitting a singleequation to the data from the article of interest combined with the datafrom the reference article.

$\begin{matrix}{{{Log}_{10}\left( y_{{ijk}\;} \right)} = {D + \frac{\left( {A - D} \right)}{1 + \left( \frac{x_{i\; j}}{C_{A}*\left( \frac{C_{R}}{C_{A}} \right)^{I}} \right)^{B}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

-   -   Where:    -   A, B and D parameters are common to both the reference and test        article and C_(R) is the reference parameter, C_(A) is the test        article parameter, and the ratio C_(R)/C_(A) is the relative        potency. The superscript I is an indicator variable. It is set        equal to 1 if the data come from the article of interest, and 0        if the data come from the CTLA4-Ig material.

The relative potency of each test article was translated to a percentagescale and the relative potency was given as output from the program.

The eight relative potency results of the output from each of the eightdata sets analyzed can be averaged and the standard deviation can becalculated using Equations 3 and 4, respectively. The average result isreported as “percent relative potency” rounded to the nearest wholenumber.

$\begin{matrix}{{Average} = \frac{\begin{matrix}{{{Value}\; 1} + {{Value}\; 2} + {{Value}\; 3} + {{Value}\; 4} +} \\{{{V{alue}}\; 5} + {{Value}\; 6} + {{Value}\; 7} + {{Value}\; 8}}\end{matrix}}{8}} & {{Equation}\mspace{14mu} 3} \\{{{Standard}\mspace{14mu} {Deviation}} = \sqrt{\frac{{8{\sum x^{2}}} - \left( {\sum x} \right)^{2}}{8\left( {8 - 1} \right)}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

-   -   Where:    -   8 is the number of potency measurements        -   x=individual measurements

Adjustment of Relative Potency Values Obtained With ApproximateConcentrations: Due to the time lag between sample receipt and obtaininga precise protein concentration, a sample may be tested in the assay atan approximate concentration and results adjusted when the preciseconcentration is determined. This adjustment is performed using Equation5 below where the relative potency determined in the assay is multipliedby the ratio of the CTLA4-Ig concentration used to set up the assay tothe determined CTLA4-Ig sample concentration.

$\begin{matrix}{{{Reportable}\mspace{20mu} {Relative}\mspace{20mu} {Potency}} = \frac{{Observed}\mspace{14mu} {Relative}\mspace{14mu} {Potency}*{Concentration}\mspace{20mu} {Used}}{{Determined}\mspace{14mu} {Concentration}}} & {{Equation}\mspace{14mu} 5}\end{matrix}$

-   -   Example:    -   Sample was tested at a protein concentration of 25 mg/mL in the        assay.    -   Relative Potency determined was 105%.    -   Determined CTLA4-Ig concentration was determined to be 25.5        mg/mL

Reportable Relative Potency=(105*25)/25.5=103%.

The test article relative potency values must be between 25 and 175% ofthe reference standard, which is the range of the assay. If the relativepotency values are outside this range, then the sample must be dilutedor concentrated in order to fall within this range and the samplereanalyzed.

Example 46 Structural Characterization of O-Linked Oligosaccharides

The O-linked glycosylation of CTLA4-Ig was characterized by peptidemapping followed by ESI-MS/MS, and by MALDI-TOF.

Analysis of T8 and T9 by Peptide Mapping with ESI-MS/MS

A modified manual version of the tryptic digestion method with in-lineESI-MS/MS using a Finnigan Ion Trap mass spectrometer was performed inorder to characterize O-linked glycopeptides T8 and T9 (refer to Table59 for peptide identity).

FIG. 62 shows the tryptic peptide map of CTLA4-Ig indicating that T8elutes at the end of the solvent front, and T9 elutes at the shoulder ofT27.

The full mass spectrum of peptide T8 is shown in FIG. 63, where peak436.2 corresponds to the expected MW of the singly-charged unmodifiedpeptide T8, and peak 1092.2 correspond to the MW of the singly-chargedglycosylated T8. The mass corresponding to the major peak and itsstructure (T8-HexNAc-Hex-NeuAc) are shown in FIG. 96.

The full mass spectrum of peptide T9 is shown in FIG. 64, wheredoubly-and triply-charged ions of the glycopeptides appear,corresponding to a range of heterogeneous glycoforms. Major peak 1115.9corresponds to the MW of the triply charged T9 glycosylated withHexNAc-Hex-NeuAc. Peaks 1213.0 and 1334.8 correspond to molecularweights of the triply-charged T9, glycosylated withHexNAc(NeuAc)-Hex-NeuAc, and (HexNAc-Hex-NeuAc)₂, respectively (FIG.97). The dominant glycoform is monosialyated T9-HexNAc-Hex-NeuAc whilethe other glycoforms are present at a much lower abundance.

In order to determine the O-linked sites, peptide mapping of CTLA4-Igwas performed as previously described in Example 4 and the fraction T8-9(due to incomplete digestion by trypsin) was collected and subjected toEdman sequencing. The sequencing data showed that in the 1st cycle, anextra peak appears in addition to Ser. The retention time of the extrapeak does not agree with that of any of the standard amino acids,suggesting that Ser at position 1 in T8 (Ser 129) is modified andcontains O-linked glycans. The appearance of both Ser and the extra peakindicate that Ser is partially modified. This is in agreement with theMS data for T8. The sequencing experiments also reveal the O-linked sitein T9 as Ser 139. In conclusion, two O-linked glycosylation sites wereidentified at amino acid residues serine 129 and serine 139. Thepredominant glycan attached to the two sites is HexNAc-Hex-NeuAc.

MALDI-TOF Analysis of T9 Peptide

MALDI-TOF analysis of peptide T9 demonstrates the presence of severalglycoforms (FIG. 65). The peak with a MW of 2690.8 is consistent withthe T9 fragment. The peak with a MW of 2893.7 correlates to T9 plusHexNAc. The peak with a MW of 3055.7 correlates to T9 plus HexNAc-Hex.The peak with a MW of 3345.8 indicates T9 plus HexNAc-Hex-NANA.Galactose and N-Acetyl Galactosamine were detected in T9 based onmonosaccharide analysis, therefore the major O-linked species in T9 ispostulated to be GalNAc-Gal-NANA. MALDI-TOF analysis of peptide T8 wasnot performed due to low recovery yields.

Example 47 Oxidation and Demidation Variants in CTLA4-Ig

Oxidation and deamidation are common product variants of peptides andproteins. They can occur during fermentation, harvest/cellclarification, purification, drug substance/drug product storage, andduring sample analysis.

Protein oxidation is typically characterized by the chemical addition ofone or more oxygen atoms to the protein. Several amino acids, Met, Cys,Tyr, His and Trp, are more susceptible to oxidation compared to othernatural amino acids. The amino acid with the highest degree ofsusceptibility to oxidation is methionine. The majority of proteinoxidations identified to date have been the oxidation of methionine tothe sulfoxide variant. Oxidation in proteins can be caused by severaldifferent mechanisms. The common mechanism of oxidation occurs fromlight exposure or transition metal catalysis.

Deamidation is the loss of NH₃ from a protein forming a succinimideintermediate that can undergo hydrolysis. The succinimide intermediateresults in a 17 u mass decrease of the parent peptide. The subsequenthydrolysis results in an 18 u mass increase. Isolation of thesuccinimide intermediate is difficult due to instability under aqueousconditions. As such, deamidation is typically detectable as 1 u massincrease. Deamidation of an asparagine results in either aspartic orisoaspartic acid. The parameters affecting the rate of deamidationinclude pH, temperature, solvent dielectric constant, ionic strength,primary sequence, local polypeptide conformation, and tertiarystructure. The amino acid residues adjacent to Asn in the peptide chainaffect deamidation rates. Gly and Ser following an Asn in proteinsequences results in a higher susceptibility to deamidation.

Materials and Methods

Sample: CTLA4-Ig Standard

Trypsin/Asp-N/Trypsin and Chymotrypsin Peptide Mapping of CTLA4-Ig:Proteins were denatured and reduced in 50 mM Tris buffer (pH 8.0)containing 6 M Guanidine and 5 mM dithiothreitol (DTT). After 20-minutesincubation at 50° C., iodoacetamide (IAM) was added to a finalconcentration of 10 mM and the sample was incubated in darkness at 50°C. for an additional 20 minutes. The reduced and alkylated mixture wasloaded onto a NAP-5 column, and then eluted out with 50 mM Tris, 10 mMCaCl₂, pH 8.0. Sequence grade trypsin (2% w/w, enzyme : protein) wasadded and incubated for 4 hours at 37° C. In the case of Asp-Ndigestion, sequence grade Asp-N (4% w/w, enzyme : protein) was added andthe sample was incubated for 16 hours at 37° C.

In the case of trypsin and chymotrypsin digestion, the protein was in 50mM sodium phosphate buffer, pH 7.5. Sequence grade trypsin (4%, w/w,enzyme : protein) was added and the sample was incubated for 4 hours at37° C. Chymotrypsin was added (4%, w/w, enzyme : protein) and the sampleincubated for 16 hours at 37° C. Samples were stored at −20° C. afterthe digestion.

Peptide mixtures were separated by gradient elution from an Atlantis C18column (2.1×250 mm) on a Waters Alliance HPLC Workstation (Waters,Milford, Mass.) at 0.120 mL/min. The column was directly connected tothe Q-Tof micro (Waters, Milford, Mass.) equipped with an electrosprayionization spray source and mass spectra were collected. In the case ofAsp-N peptide mapping, peptide mixtures were separated on a Varian C18column (4.6×250 mm) at 0.7 mL/min using the same HPLC workstation. Thecolumns were equilibrated with solvent A (0.02% TFA in water) andpeptides were eluted by increasing concentration of solvent B (95%Acetonitrile/0.02% TFA in water). A post-column splitter valve was usedto direct 15% of the flow to the Q-Tof micro. The instrument was run inthe positive mode (m/z 100-2000). The capillary voltage was set to 3000V.

MS/MS Analysis of Peptides: Fractions from reversed phase chromatographywere collected and infused into the Q-Tof micro at a flow rate of 20μL/minute. The MS/MS spectra were acquired using a collision energyoptimized for the individual peptide (ranging from 25 to 42 eV).

Results

Oxidation of CTLA4-Ig: CTLA4-Ig has seven methionines per single chain:Met', Met⁵³, Met⁵⁴, Met⁸⁵, Met⁹⁷, Met^('62) and Met³³⁸. Peptide mappingwas used to identify the oxidative product variants at each of thesesites. Oxidations of Met', Met⁸⁵ and Met'⁶² are identified using thetryptic peptide mapping technique (FIGS. 66A and 66B). No oxidation ofMet³³⁸ is detected. The tryptic fragments containing Met⁵³, Met⁵⁴ orMet⁹⁷ are large peptides containing heterogeneous N-linkedcarbohydrates. These peptides are not amenable to identification andrelative quantitation of oxidation. Therefore, proteolysis usingmultiple enzymes, which produce shorter, non-glycosylated peptides wasperformed. The Asp-N peptide EVCAATYMMGN (46-56) andtryptic/chymotryptic peptide MYPPPY (97-102) are used to determineoxidation of Met⁵³, Met⁵⁴ and Met⁹⁷. The relative quantitation ofmethionine oxidation is calculated according to the peak area percent ofthe extracted ion chromatograms. The relative amounts of oxidizedpeptides are listed in Table 63. Oxidation on six out of the sevenCTLA4-Ig methionines per single chain is detected. Peaks A and B are thesingly oxidized forms of peptide EVCAATYMMGN (46-56) (peak 3). Peptidesfrom peaks 2A and 2B are isobaric and have a mass increase of 16 u ascompared with the unmodified peptide mass. Each peak representsoxidation at different Met. The degree of the oxidation is different atthe two sites.

TABLE 63 The Oxidation of Met in CTLA4-Ig Expected Observed Mass MassExpected Observed Non- Non- Mass Mass Oxidation Met Peptide oxidizedoxidized Oxidized Oxidized Percent 1 AT1: AMHVAQPAVVLASSR  768.9 (+2)768.9 776.91 (+2) 776.9 0.9 1 T1: MHVAQPAVVLASSR  733.4 (+2) 733.4 741.4 (+2) 741.4 2.4 53 D5: EVCAATYMMGN 1246.5 1246.6 1262.5 1262.5 3.454 D5: EVCAATYMMGN 1246.5 1246.6 1262.5 1262.6 4.3 85 T6: AMDTGLYICK1171.6 1171.5 1187.6 1187.5 1.0 97 MYPPPY 767.3 767.9 783.3 783.9 1.5162 T10: DTLMISR 835.4 835.4 851.4 851.4 1.7 338 T30: 1401.1 (+2) 1401.11409.1 (+2) WQQGDVFSCSVMHEALHNHYTQK *Due to the minor contribution ofAT1 oxidation, the percent of AT1 oxidation is not included in thecalculation of the estimated amount of the CTLA4-Ig oxidation.

The total estimated amount of CTLA4-Ig methionine oxidation iscalculated to be 2.0% for CTLA4-Ig. This is calculated by adding thetotal percentage of oxidation at each site and dividing by the totalnumber of methionines, which is seven. MS/MS analysis was performed onoxidized and native peptides listed in Table 63.

All peptides, with the exception of D5, are sequenced using MS/MS. Theoxidized amino acid is determined by the mass difference within the band y ion series. The MS/MS spectra of the oxidized and native T1peptide entails the doubly charged precursor ions at m/z 741.4 and733.4. The mass difference is 8 u (for the double charge state) that is16 u when corrected for the charge state. The tryptic peptide T1 (1-14)contains the Met¹ residue. The native peptide and its oxidizedderivative are baseline separated by reversed phase chromatography. Theion chromatogram for doubly charged ions of T1 and its derivatives isshown in FIGS. 66A and 66B. The b and y ion series are the predominantions produced in collision induced dissociation. y6-y13 ions have thesame modified and unmodified ion masses. The b2 ion of the oxidizedpeptide is 16 u higher than the corresponding b2 ion of the nativepeptide. The b and y ion series taken together with the peptide massesidentify methionine 1 as the amino acid modified in the T1 peptide. Inthe same way, Met oxidations in AT1, T6, T10, and MYPPPY (97-102)peptides are identified.

Deamidation of CTLA4-Ig: CTLA4-Ig has 15 asparagines per single chain.Three asparagines are known to be attached to N-linked carbohydratestructures. Peptide mapping was used to identify and relatively quantifydeamidation occurring at the other 12 Asn residues. Deamidations ofAsn^(186,) Asn²²⁵, Asn²⁷¹, Asn²⁹⁴ and Asn³⁴⁴ are identified by the LC/MStryptic peptide map (Table 64). The relative quantitation of asparaginedeamidation is calculated according to the peak area percent of theextracted ion chromatograms. The relative amounts of deamidated peptidesare listed in Table 64. The total estimated amount of CTLA4-Igasparagine deamidation is calculated to be 0.3% for CTLA4-Ig. This iscalculated by adding the total percentage of deamidation at each siteand dividing by 15. MS/MS analysis was performed on deamidated andnative peptides listed in Table 64.

TABLE 64 The Deamidation of Asn in CTLA4-Ig Observed Expected MassExpected Observed Mass Non- Non- Mass Mass Deamidation Asn Peptidedeamidated deamidated Deamidated Deamidated Percent 186 T12:  839.4 (+2)839.4  839.9 (+2) 839.9 0.9 FNWYVDGVEVHNAK 225 T15:  904.5 (+2) 904.5 905.0 (+2) 905.0/ 1.5/0.8 VVSVLTVLHQDWLN 905.0 GK 271 T25: NQVSLTCLVK1161.6 1161.7 1162.6 1162.7 0.3 294 T26: 1272.6 (+2) 1272.6 1273.1 (+2)1273.1 1.2 GFYPSDIAVEWESNG QPENNYK 344 T30: 1401.1 (+2) 1401.2 1401.6(+2) 1401.7 0.2 WQQGNVFSCSVMHE ALHNHYTQK

The deamidated amino acid is determined by the mass difference withinthe b and y ion series. For example, if there are two deamidated peaksfor peptide T15—masses 905.0 u. Peak 1 elutes prior to the native peak;peak 3 elutes after the native peak. Tryptic peptides and theirdeamidated forms are baseline separated on the reversed phase column.MS/MS analysis was performed on tryptic peptides and deamidated peptidesare listed in Table 64. Peaks 1-3 contain the same y1 and y2 ions,indicating that the C-terminal amino acids are the same for all threepeaks; however, the y3-y14 ions from peaks 1 and 3 are 1 u higher thanthe corresponding ions from peak 2. The mass difference between y2 andy3 is 114 u for peak 2; this corresponds to an Asn residue. The 115 ufor peaks 1 and 3 is a 1 u mass increase compared to the Asn residuemass; this corresponds to either aspartic or isoaspartic acid. In thesame way, deamidations in T12, T25, T26 and T30 were identified and thefragment ions identify the modification sites.

Asparagine deamidations and methionine oxidations are determined fromLC/MS and LC/MS/MS analyses of the endopeptidase cleavage of CTLA4-Ig.The modifications are identified by shifts in masses between themodified and unmodified peptides. The modified amino acids areidentified by MS/MS sequencing. Six methionine oxidations and fiveasparagine deamidations per single chain are present in CTLA4-Igmaterial in a small percentage. CTLA4-Ig Met¹, Met⁵³, Met⁵⁴, Met⁸⁵,Met⁹⁷, and Met¹⁶² are all found to be oxidized. These oxidationsdetermined from CTLA4-Ig are found to be less than 2.5% of all CTLA4-Igmethionines. CTLA4-Ig Asn^(186,) Asn²²⁵, Asn²⁷¹, Asn²⁹⁴, and Asn³⁴⁴ areall found to be deamidated in low amounts. These deamidations determinedfrom CTLA4-Ig are found to be less than 0.5% of all CTLA4-Igasparagines.

Example 48 Bacterial Endotoxin Assays for CTLA4-Ig andCTLA^(4A29YL104E)-Ig

In one embodiment, the CTLA4-Ig composition drug substance has less thanor equal to 0.15 EU/mg bacterial endotoxin. CTLA4-Ig is assayed for thepresence of bacterial endotoxins using the Limulus Amebocyte Lysate(LAL) gel clot technique based on USP<85>. In preparing for and applyingthe assay, observe precautions in handling the specimens in order toavoid gross microbial contamination; treat any containers or utensilsemployed to destroy extraneous endotoxins that may be present on theirsurfaces such as heating in a dry-heat oven using a validated cycle.

LAL Reagent: (Associates of Cape Cod, Inc.-or equivalent) LyophilizedLimulus

Amebocyte Lysate (LAL) reagent such as Pyrotell should be storedaccording to manufacturer's instructions. Reconstituted LAL reagent maybe held frozen for no longer than one month, and should not be thawed orfrozen more than once.

Endotoxin Standards: (Associates of Cape Cod, Inc.- or equivalent) Thecontrol standard endotoxin (CSE) used in the tests must be traceable andstandardized against Reference Standard Endotoxin (RSE). Storeunreconstituted containers of endotoxin in a refrigerator; oncereconstituted, they may be held at 2°-8° C. for no longer than 14 days,unless validation for longer periods shows suitable reactivity.

Testing of Production Lots

The lack of product inhibition or enhancement of the LAL procedureshould be validated for each drug formulation. Testing of productionlots is performed using three individual units representing beginning,middle, and end of production. These units can be run individually orpooled. A representative positive product control of the sample at thetest concentration, inoculated with twice the amount of CSE (or RSE) asthe labeled sensitivity of the lysate, must be included for a validtest. Adjust the pH so that the final product/lysate solution is in therange of 6.0 to 8.0, using sterile endotoxin-free HCl, NaOH or suitablebuffer. In some situations, it may be helpful to use a buffering agentsuch as Pyrosol as an alternative to pH adjustment. Refer to themanufacturer's directions for specific use.

Use of Buffering Agent for Lysate Reconstitution

A buffering agent such as Pyrosol (Associates of Cape Cod, Inc.) issusedto reconstitute the lysate used for testing and is designed to amplifythe buffering capacity of the lysate. Pyrosol-reconstituted lysate maybe used to test samples or sample dilutions that may otherwise requireadjustment of pH with acid or base or which precipitate on adjustment ofpH. When combined with the test sample, Pyrosol-reconstituted lysategives a pink color to indicate that the pH is in range for a valid test.If outside that range, the color will be yellow or purple; in this case,the test sample would require additional pH adjustment. Specific methodswill dictate the use of Pyrosol for lysate reconstitution.

Use of Glucan-Inhibiting Buffer for Lysate Reconstitution

A glucan-inhibiting buffer such as Glucashield (Associates of Cape Cod,Inc.) is used to reconstitute the lysate used for testing and isdesigned to block potential glucan interference.Glucashield-reconstituted lysate may be used to test samples or sampledilutions that may contain glucan contamination. Interference fromglucan contamination can give false positives in certain test materials,so using a Glucashield-reconstituted lysate may block or reduceinterference allowing for a reduced number of false positives. Specificmethods will dictate the use of Glucashield for lysate reconstitution.

Sample Dilutions

If necessary to approximate the level of endotoxin concentration in thesample, prepare an appropriate series of dilutions of the sample insterile endotoxin-free water. Vortex and add 0.1 mL of each preparationto be tested to each of two sterile endotoxin-free 10×75 mm glass testtubes.

Preparation of Endotoxin Standard Solutions for Standard Curve

Reconstitute the vial of endotoxin with endotoxin-free water as per themanufacturer's instructions. Mix the vial vigorously on a vortex mixerintermittently for 30 minutes. Preserve the concentrate in arefrigerator at 2°-8° C. for no more than 4 weeks. Mix vigorously usinga vortex mixer for not less than 3 minutes prior to use. Consult themanufacturer's Certificate of Analysis to verify the concentration ofthe endotoxin stock, and prepare a standard endotoxin dilution series,designed to bracket the endpoint, such as in the following example:

-   0.5 mL (1000 EU/mL)+9.5 mL endotoxin-free water=50 EU/mL-   5.0 mL (50 EU/mL)+5.0 mL endotoxin-free water=25 EU/mL-   1.0 mL (25 EU/mL)+9.0 mL endotoxin-free water=2.5 EU/mL-   1.0 mL (2.5 EU/mL)+9.0 mL endotoxin-free water=0.25 EU/mL-   5.0 mL (0.25 EU/mL)+5.0 mL endotoxin-free water=0.125 EU/mL-   5.0 mL (0.125 EU/mL)+5.0 mL endotoxin-free water=0.06 EU/mL-   5.0 mL (0.06 EU/mL)+5.0 mL endotoxin-free water=0.03 EU/mL-   5.0 mL (0.03 EU/mL)+5.0 mL endotoxin-free water=0.015 EU/mL

Be sure to vigorously vortex each dilution for at least 30 secondsbefore proceeding to the next dilution. Include a negative water controlof the diluent used. Vortex and add 0.1 mL of each of the 0.125 EU/mL,0.06 EU/mL, 0.03 EU/mL, 0.015 EU/mL concentrations (or alternate curve)and the negative water control, to each of two sterile endotoxin-free10×75 mm glass test tubes to provide two replicate curves.

Preparation of LAL Reagent Solution

Remove the lyophilized LAL reagent from the freezer. Aseptically add 5.0mL of endotoxin-free water (unless otherwise directed by a specificmethod to use a reconstitution buffer) to the vial. Swirl or roll thevial to dissolve the reagent.

Preparation of Positive Product Control

All products must be tested with a positive product control. Refer tothe applicable Specific Method for instructions on preparation of thepositive control. If no instruction is provided, prepare the positiveproduct control to contain an endotoxin spike at 2× the level of thelysate sensitivity, in combination with the product at its test level.When testing Water for Injection or High Quality Process Water, use adilution of the reconstituted endotoxin standard so that when added tothe sample, the endotoxin concentration will be 2× the LAL solutionsensitivity. For example, if the lysate sensitivity=0.06 EU/mL, add 0.1mL of a 2.5 EU/mL endotoxin solution to 1.9 mL of test sample (in theform as added to the LAL solution)=0.125 EU/mL. The volume of theendotoxin solution is not greater than 0.1 mL and the overall dilutionis not less than 1:20.

Test Procedure

1. Aseptically dispense 0.1 mL of the LAL reagent solution into each ofthe test sample tubes and endotoxin standard tubes, being cautious notto crosscontaminate between tubes. NOTE: Place any remaining reagent ina freezer at about minus 10° C. to minus 25° C.

2. Refer to the specific lysate insert for mixing instructions of theproduct-lysate mixture.

3. Place each tube in an incubating device such as a water bath orheating block maintained at 37° ±1° C. Record the incubation start timeand the temperature of the water bath or heating block.

4. Incubate each tube, undisturbed, for 60±2 minutes.

5. Following the incubation, record the incubation end time and observeeach tube by gently inverting the tube 180° . A positive result isdenoted by a firm gel which remains firm when inverted carefully, anegative result is characterized by the absence of such a gel, or by theformation of a viscous gel that does not maintain its integrity. Handlethe tubes carefully and avoid subjecting them to vibrations which couldcause false negative observations.

Evaluation

The lowest concentration in each replicate series to give a positiveresult is called the endpoint. Calculate the geometric mean endpoint forthe test by the following procedure: Geometric Mean=The antilogarithm ofthe logarithmic sum of the gelation endpoints divided by the number ofreplicate endpoint assays. The test is valid provided the geometric meanendpoint is within a two-fold dilution of the labeled lysatesensitivity, the positive product control is positive, and the negativewater control is negative. Determine the approximate bacterial endotoxinlevel in or on the test item by comparing the labeled lysate sensitivitywith the positive or negative results coupled with the dilution factorsof the item tested. The article meets the requirements of the test ifthere is no formation of a firm gel at the level of endotoxin specifiedin the individual monographs or specification.

Method for CTLA4^(A29YL104E)-Ig

This method is used to quantify bacterial endotoxins in samples ofCTLA4^(A29YL104E)-Ig drug substance and drug product using the kineticturbidimetric LAL method. The results are reported as EU/mL and EU/mgdrug product equivalent.

Terms:

LAL—Limulus Amebocyte Lysate

CSE—Control Standard Endotoxin

EU—United States Pharmacopoeia Endotoxin Units

EU/mg—United States Pharmacopoeia Endotoxin Units per milligram

EU/mL—United States Pharmacopoeia Endotoxin Units per milliliter

LRW—LAL Reagent Water

Limulus Amebocyte Lysate Turbidimetric (PYROTELL-T®) Solution. Allowvials of PYROTELL-T® and PYROSOL® to warm to room temperature for 30minutes before opening. Remove metal seal from PYROTELL-T® andaseptically remove stopper. Reconstitute PYROTELL-T® with 5.0 mLPYROSOL® Reconstitution Buffer. Swirl bottle gently to mix untilcompletely dissolved. Reconstitute buffer immediately before use.Reconstituted PYROTELL-T® can be kept at 2-8° C. for no more than 24hours. Cover the top of container with a layer of PARAFILM®.

Control Standard Endotoxin Solution. Allow vial of CSE (1.2) to warm toroom temperature for 30 minutes before opening. Remove metal seal fromthe vial and aseptically remove stopper. Carefully lift the stopper justenough to allow air to enter, thereby breaking the vacuum. ReconstituteControl Standard Endotoxin (CSE) with 5.0 mL of LRW (1.4). Seal withPARAFILM®. Vortex vigorously for one minute, at 5-10 minute intervalsover a 30-60 minute interval at room temperature. CSE is then ready foruse. Refer to the manufacture's Certificate of Analysis to obtain theControl Standard Endotoxin (CSE) potency in USP-EU per vial vs. thespecific lot of PYROTELL-T®. Calculate the USP-EU/mL of the CSE in thevial. Reconstituted CSE can be kept at 2-8° C. for 30 days. After eachuse, seal container with new PARAFILM®.

-   -   Example:    -   For a vial having a potency of 6,000 USP-EU/vial reconstituted        with 5.0 mL of LRW, the potency would be 1,200 USP-EU/mL.

${Potency} = {\frac{6,000\mspace{14mu} {EU}\text{/}{vial}}{5.0\mspace{14mu} {mL}} = {1,200\mspace{14mu} {EU}\text{/}{mL}}}$

Set Up Assay Parameters in PYROSOFT-11® Software. General Parameters.Set up parameters in the General Test Information Tab as shown in thebelow Table.

General Test Information

Parameter Value Valid Temp Range Min. 36.5 Valid Temp Range Max. 37.5Range for Spike Recovery Min. 50 Range for Spike Recovery Max. 200Threshold OD (mAbs) 20 Max OD Stored (mAbs) 100 Maximum Test Time (mins)120 Baseline Adj. Active Check Auto-end Uncheck Check negative controlUncheck

Options

Parameter Value Flag Correlation Coefficient Checked Display CorrelationCoefficient Not checked Flag Coefficient of Variation Not checkedExtrapolate beyond Not checked Auto Test ID Not checked Show Pass FailResults Checked Auto-Print Not checked

Tube Assignments-Example

Tube Sample Standard/Spike Sample No. Description Concentration UnitsDilution* 1 Negative Control EU/mL 2 Negative Control EU/mL 3 Standard0.064 EU/mL 4 Standard 0.064 EU/mL 5 Standard 0.032 EU/mL 6 Standard0.032 EU/mL 7 Standard 0.016 EU/mL 8 Standard 0.016 EU/mL 9 Standard0.008 EU/mL 10 Standard 0.008 EU/mL 11 Standard 0.004 EU/mL 12 Standard0.004 EU/mL 13 Standard 0.002 EU/mL 14 Standard 0.002 EU/mL 15 Sample 1EU/mL 1:1 16 Sample 1 EU/mL 1:1 17 Sample 1 Spike 0.016 EU/mL 1:1 18Sample 1 Spike 0.016 EU/mL 1:1 19 Sample 2 EU/mL 1:1 20 Sample 2 EU/mL1:1 21 Sample 2 Spike 0.016 EU/mL 1:1 22 Sample 2 Spike 0.016 EU/mL 1:123 Sample 3 EU/mL 1:1 24 Sample 3 EU/mL 1:1 25 Sample 3 Spike 0.016EU/mL 1:1 26 Sample 3 Spike 0.016 EU/mL 1:1 27 Sample 4 EU/mL 1:1 28Sample 4 EU/mL 1:1 29 Sample 4 Spike 0.016 EU/mL 1:1 30 Sample 4 Spike0.016 EU/mL 1:1 31 Positive Water Control 0.016 EU/mL 32 Positive WaterControl 0.016 EU/mL

Prepare Standard Curve Concentrations. Prepare Control StandardEndotoxin (CSE, 2.2) working stock solution at 4 USP-EU/mL. Prepare aworking solution of CSE at a potency of 4 EU/mL. Calculate the volume ofLRW needed for the dilution using Equation 1. Add 20 μL of CSE solution(2.2) to the appropriate amount (Equation 1) of LRW (1.4) in apolystyrene tube (1.9). Vortex dilution for 30 seconds.

-   -   Example:    -   For CSE solution at a potency of 1,200 EU/mL add 20 μL of CSE        solution to 5,980 μL of LRW.

${LRWVolume} = {\left( \frac{20\mspace{14mu} {µL}*1,200\mspace{14mu} {EU}\text{/}{mL}}{4\mspace{14mu} {EU}\text{/}{mL}} \right) - {20\mspace{14mu} {µL}}}$

Prepare CSE working stock solution at 0.64 EU/mL. Prepare a workingsolution of CSE at a potency of 0.64 USP-EU/mL by adding 1.6 mL of CSEstock solution at 4 USP-EU/mL to 8.4 mL of LRW in a polystyrene tube.Vortex for 30 seconds.

Prepare Standard Stock Solutions. Standard Stock Solutions are preparedat 0.128, 0.064, 0.032, 0.016, 0.008, and 0.004 EU/mL from the workingsolution of CSE in polystyrene tubes. Vortex each tube for 30 secondsafter dilution. A dilution scheme is shown in the Table below.

Dilution Scheme for Standard Stock Solutions Final Stock Volume (μL) ofConcentration Concentration (EU/mL) LRW (mL) (EU/mL)* 2 mL of 0.64 EU/mLsolution 8 0.128 4 mL of 0.128 EU/mL solution 4 0.064 4 mL of 0.064EU/mL solution 4 0.032 4 mL of 0.032 EU/mL solution 4 0.016 4 mL of0.016 EU/mL solution 4 0.008 4 mL of 0.008 EU/mL solution 4 0.004 *Thefinal two-fold dilution occurs in the reaction tubes.

Sample Preparation. Drug Substance Sample Preparation. Prepare a sampledilution to 0.25 mg/mL. Calculate the volume of LRW needed for thedilution using the Equation below. Carefully remove top of samplecontainer and add 50 μL of sample to the appropriate amount (Equationbelow) of LRW in a polystyrene tube to make a 0.25 mg/mL solution.Vortex sample for 30 seconds. Cover the top of sample container withlayer of PARAFILM®.

-   -   Example:    -   For a sample having a concentration of 24.7 mg/mL add 50 μL        (0.05 mL) of sample to 4.89 mL of water

$\begin{matrix}{{L\; R\; W\mspace{14mu} {Volume}\mspace{14mu} ({mL})} = {\left( \frac{0.05*{Protein}\mspace{14mu} {Concentration}\mspace{14mu} \left( {{mg}\text{/}{mL}} \right)}{0.25\mspace{14mu} {mg}\text{/}{mL}} \right) - {0.05\mspace{14mu} {mL}}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Drug Product Lyophile Sample Preparation. NOTE: A single Drug Productlot consists of three separate samples, “beginning”, “middle”, and“end”. Reconstitute a vial of drug product with LAL Reagent wateraccording to the Product Identification (PI) specifications. Dilute thereconstituted sample to 0.25 mg/mL. Calculate the volume of LRW neededfor the dilution using Equation 2. Carefully remove top of samplecontainer and add 50 μL of sample to the appropriate amount (Equation 2)of LRW in a polystyrene tube to make a 0.25 mg/mL solution. Vortexsample for 30 seconds. Cover the top of sample container with PARAFILM®.

Drug Product Ready-to-Use Sample Preparation. Dilute the sample to 0.25mg/mL. Calculate the volume of LRW needed for the dilution usingEquation 3. Carefully remove top of sample container and add 10 μL ofsample to the appropriate amount (Equation 3) of LRW in a polystyrenetube to make a 0.25 mg/mL solution. Vortex sample for 30 seconds. Coverthe top of sample container with PARAFILM®.

-   -   Example:    -   For a sample having a concentration of 125.0 mg/mL add 10 μL        (0.01 mL) of sample to 4.99 mL of water

$\begin{matrix}{{L\; R\; W\mspace{14mu} {Volume}\mspace{14mu} ({mL})} = {\left( \frac{0.01*{Protein}\mspace{14mu} {Concentration}\mspace{14mu} \left( {{mg}\text{/}{mL}} \right)}{0.25\mspace{14mu} {mg}\text{/}{mL}} \right) - {0.01\mspace{14mu} {mL}}}} & {{Equation}\mspace{14mu} 3}\end{matrix}$

Drug Product Ready-to-Use Placebo. Dilute the placebo (as if it were ata nominal drug product protein concentration of 125 mg/mL) to 0.25mg/mL. This is equivalent to a 1:500 dilution. Calculate the volume ofLRW needed for the dilution using Equation 3. Carefully remove top ofsample container and add 10 μL of sample to the appropriate amount(Equation 3) of LRW in a polystyrene tube to make an 0.25 mg/mLequivalent solution.

Positive Water Control. The positive control is prepared from thestandard curve by adding 100 μL of 0.032 EU/mL standard to 100 μL of LRWin the reaction tubes.

Reaction Tube Setup—Example

Standard Standard/ Spike Final Tube Stock Conc. Samples LRW Soln. Conc.No. Description (EU/mL) (μL) (μL) (μL) (EU/mL) 1 Negative Control — 0200 0 0.000 2 Negative Control — 0 200 0 0.000 3 Std. 1 0.004 100 100 00.002 4 Std. 1 0.004 100 100 0 0.002 5 Std. 2 0.008 100 100 0 0.004 6Std. 2 0.008 100 100 0 0.004 7 Std. 3 0.016 100 100 0 0.008 8 Std. 30.016 100 100 0 0.008 9 Std. 4 0.032 100 100 0 0.016 10 Std. 4 0.032 100100 0 0.016 11 Std. 5 0.064 100 100 0 0.032 12 Std. 5 0.064 100 100 00.032 13 Std. 6 0.128 100 100 0 0.064 14 Std. 6 0.128 100 100 0 0.064 15Sample 1 — 100 100 0 unknown 16 Sample 1 — 100 100 0 unknown 17 Sample 1Spike — 100 0 100 Constitutive + 0.016 18 Sample 1 Spike — 100 0 100Constitutive + 0.016 19 Sample 2 — 100 100 0 unknown 20 Sample 2 — 100100 0 unknown 21 Sample 2 Spike — 100 0 100 Constitutive + 0.016 22Sample 2 Spike — 100 0 100 Constitutive + 0.016 23 Sample 3 — 100 100 0unknown 24 Sample 3 — 100 100 0 unknown 25 Sample 3 Spike — 100 0 100Constitutive + 0.016 26 Sample 3 Spike — 100 0 100 Constitutive + 0.01627 Sample 4 — 100 100 0 unknown 28 Sample 4 — 100 100 0 unknown 29Sample 4 Spike — 100 0 100 Constitutive + 0.016 30 Sample 4 Spike — 1000 100 Constitutive + 0.016 31 Positive Water 0.032 0 100 100 0.016Control 32 Positive Water 0.032 0 100 100 0.016 Control

Add PYROTELL-T® Reagent. Add 50 μL of PYROTELL-T® solution to eachreaction tube (negative control, standards, samples, spiked samples, andpositive water control) with a repeat pipetor. Vortex each tube for 1-2seconds, and insert it in the assigned well (Table 1) in the PyrosKinetix instrument.

Data Analysis

Analysis. The PYROSOFT®-11 software will perform an autoanalysis of allstandards, controls, and samples and report sample results in EU/mL atthe completion of the run. Each duplicate sample is reported as a meanvalue of the two. If one of the tubes of a duplicate pair value isundetected by PYROSOFT®-11 software, that tube may be excluded fromfurther data analysis, and the results recalculated. Spike recovery(positive sample control) will be calculated based on the amount ofendotoxin in the sample plus the amount of endotoxin spiked into thesample (0.016 EU/mL).

Convert EU/mL value to EU/mg value of Diluted Drug Substance or DrugProduct Sample

-   -   Raw data is generated as EU/mL and reported as EU/mg of protein.        To convert EU/mL to EU/mg, divide the endotoxin value (EU/mL) by        the protein concentration (0.125 mg/mL). Results are reported to        one significant figure.    -   Example:    -   For a sample having 0.23 EU/mL in the assay, the reportable        EU/mg value will be 2 EU/mg (1.8 rounded to one significant        figure).

$\left( \frac{0.23\mspace{14mu} {EU}\text{/}{mL}}{0.125\mspace{14mu} {mg}\text{/}{mL}} \right) = {{1.84\mspace{14mu} {EU}\text{/}{mg}} = {2\mspace{14mu} {EU}\text{/}{mg}}}$

Result for Drug Substance Sample. The result is determined to onesignificant figure. The QL of the assay is 0.02 EU/mg. If the sample is≤0.02 EU/mg the reportable result is [<QL, (QL=0.02 EU/mg)]. Result forDrug Product Sample. The mean of the three samples for each lot of drugproduct (“beginning”, “middle”, and “end”) in EU/mg is the reportableresult for drug product samples at one significant figure. For the casewhere one or more of the samples for a lot are <QL (0.02 EU/mg), thevalue of 0.02 EU/mg will be used for calculating the mean. If the meanreportable result is ≤0.02 EU/mg the reportable result is [<QL, (QL=0.02EU/mg)].

Example:

Sample From a Lot of Drug Product Value EU/mg Beginning <QL = 0.02 EU/mgMiddle 0.023 End 0.031 Reportable Value (Mean) 0.02 EU/mg

For this example, the drug product placebo would be reported as: 3EU/mL, equivalent to 0.02 EU/mg at a nominal drug product concentrationof 125 mg/mL.

System Suitability. Drug Substance samples should be received inpolystyrene containers at 2-8° C. If samples are received in differentcontainers at a different temperature, they may not be used in theassay. The coefficient of determination for the standard curve (r²) mustbe ≥0.99. If the r² value is <0.99, the assay is not valid and must berepeated. The mean measured endotoxin concentration for the negativecontrol must be <0.002 EU/mL. If the negative control is ≥0.002 EU/mL,the assay is not valid and must be repeated. The spiked sample valuemust fall within the range of 50-200% of the expected value. If thespiked sample value is ≤49% or ≥201% of the expected value, the assay isnot valid and must be repeated. The mean measured endotoxinconcentration for the positive water control must fall within the rangeof 50-200% of the same concentration in the standard curve. If thepositive water control value is ≤49% or ≥201% of the expected value, theassay is not valid and must be repeated. Endotoxin values for thesamples must fall within the range of the endotoxin standard curve(between 0.002 and 0.064 USP-EU/mL). If samples are <0.002 EU/mL, theyare below the QL of the assay and reported per section 5.4. If samplesare >0.064 USP-EU/mL, the samples must be further diluted into the rangeof the assay.

Example 49 Microbial Limits Test (Bioburden) for CTLA4-Ig

This method provides test procedures for the estimation of the number ofviable aerobic microorganisms present and for freedom from designatedmicrobial species. In one embodiment, the CTLA4-Ig composition shouldhave bioburden at a level of CFU/10 mL. In preparing for and in applyingthe tests, observe aseptic precautions in handling the specimens. Theterm “Growth” is defined as the presence and presumed proliferation ofviable microorganisms. The validity of the test results rest largelyupon demonstration that the test specimens to which they are applied donot, of themselves, inhibit the growth, under the test conditions, ofmicroorganisms that may be present. This demonstration should includechallenging appropriately prepared specimens of the material to betested with separate viable cultures of appropriate challenge organismsto assure that the test will not inhibit growth of these clases oforganism, should they be present in the material tested. That portion ofany beta-lactam product which is used for testing must be treated withsuitable amount of penicillinase in accordance with the validatedprocedures.

Diluting Fluids and Media

1. Preparation of Culture Media and Diluting Fluids. Culture media anddiluting fluids may be prepared, or dehydrated culture media may be usedprovided that, when reconstituted as directed by the manufacturer ordistributor, they have similar ingredients and/or yield media comparableto those obtained from the formulae given herein. In preparing media bythe formulae, dissolve the soluble solids in the water, using heat ifnecessary, to effect complete solution, and add solutions ofhydrochloric acid or sodium hydroxide in quantities sufficient to yieldthe desired pH in the medium when it is ready for use. Media are to besterilized in an autoclave using a validated sterilization procedure.Determine the pH after sterilization at 25° ±2° C.

Growth Promotion

a) Each batch of autoclaved medium is tested for its growth promotingability by inoculating duplicated test containers of each medium withless than 100 microorganisms and incubating according to the conditionsspecified below. Organisms may not be more than 5 passages from theculture originally received from ATCC.

b) The test medium is satisfactory if evidence of growth appears within48-72 hours for bacteria and 5 days for fungi. The growth promotion testmay be conducted simultaneously with the use of the test medium formaterials testing. However, the materials testing is considered invalidif this growth promotion test is not successful.

Test Microorganisms for Use in Growth Promotion Testing of MediaINCUBATION TEST MEDIUM TEST MICROORGANISMS ATCC# TEMPERATURE TotalAerobic TSA (1) Staphylococcus aureus 6538 30°-35° C. Microbial Countor: Micrococcus luteus 9341 30°-35° C. (2) Bacillus subtilis 663330°-35° C. (3) Escherichia coli 8739 30°-35° C. Total Combined SDA (1)Candida albicans 10231 20°-25° C. Molds and Yeasts (2) Aspergillus niger16404 20°-25° C. Count Absence of TSB (1) Pseudomonas aeruginosa 902730°-35° C. Pseudomonas aeruginosa Absence of TSB (1) Staphyloccocusaureus 6538 30°-35° C. Staphylococcus aureus Absence of FLM (1)Salmonella choleraesuis 13311 30°-35° C. Salmonella or: Salmonellatyphimurium* 30°-35° C. Absence of FLM (1) Escherichia coli 8739 30°-35°C. Escherichia coli *Other non-pathogenic Salmonella species may also besuitable.

Procedure for Total Aerobic Microbial Count or Total Combined Molds andYeast Count

a) Sample Preparation. Unless otherwise directed by the Specific Method,prepare the specimen for testing as follows. Refer to the appropriatesection below for additional information regarding media and incubationprocedures depending on which test is being performed.

i) Bulk Powders and Raw Materials—Dissolve or suspend 10 grams or 10 mLof sample in 90 mL of sterile Phosphate Buffer pH 7.2. Mix well andtransfer 1.0 mL to each of two sterile petri dishes.

ii) Capsules—Aseptically transfer 2 capsule shells and their contents to20 mL of sterile Phosphate Buffer pH 7.2. Warm in a water bath(approximately 45° C.) for about 10 minutes. Shake vigorously until thesuspension becomes uniform, and transfer 0 mL to each of two sterilepetri dishes.

iii) Powders for suspension—Reconstitute the sample according to thelabel directions using sterile Phosphate Buffer pH 7.2 as the diluent.Shake well and transfer 1.0 mL to each of two sterile petri dishes.

iv) Solutions/Suspensions—Transfer 1.0 mL to each of two sterile petridishes.

v) Tablets—Transfer 4 tablets (hard tablets should first be pulverizedwith a sterile mortar and pestle) to 20 mL of sterile Phosphate BufferpH 7.2. Shake well until the tablets completely disintegrate ordissolve, and transfer 1.0 mL to each of two sterile petri dishes.

vi) Capsule Shells—Transfer 25 capsule shells to 100 mL SterilePhosphate Buffer pH 7.2 and warm in a water bath (approximately45° C.)for about 15 minutes, shaking intermittently to dissolve. Shake well andtransfer 1.0 mL to each of two sterile petri dishes.

vii) Ointments—Into a sterile container, pool approximately 5 mL fromeach of 3 samples taken from across the lot and mix. Transfer 0.1 mL ofthis pooled sample to each of ten petri dishes. Spread the sample overthe media surface with the aid of a sterile bent glass rod (hockeystick). Using a separate sterile rod for each plate incubate asdescribed in “Total Aerobic Count.”

b) Membrane Filtration. As an alternative to pour plating procedures, asuitable, validated membrane filtration test procedure may be used. Thismay be especially useful for products containing inhibitory substances.

c) Retesting. For the purpose of confirming a doubtful result by any ofthe following procedures, a retest may be performed using two andone-half times(minimum) the initial sample size, with appropriatediluent adjustments.

Test for Total Aerobic Microbial Count

1. Prepare the samples to be tested as described. To each plate, add15-20 mL of sterile TSA which has been melted and cooled to aproximately45° C. Cover each dish, and gently tilt or swirl the dish to mix thesample with the agar and allow the contents to solidify at roomtemperature. Invert the plates and incubate for 48 to 72 hours at30°-35° C.

2. Following incubation, examine the plates for growth using amagnification device such as a Quebec Colony Counter, count the numberof colonies and calculate the results on a unit basis (per tablet,capsule, mL, gram, etc.) as designated in the materials specificationand evaluate for acceptability against the materials specification.

3. Further characterize bacterial contamination by gram stain andmicroscopic morphology. Subject gram-negative bacteria and gram-positivecocci to biochemical testing (or alternate suitable means ofidentification).

Note: When counting colonies, in order to facilitate differentiation ofcolonial growth from the material being tested, it is at times advisableto use a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) as anenhancing agent for observing microbial growth. TTC is a colorlessoxidation-reduction indicator that turns red when it is hydrogenated byreducing sugars found in living cells, thereby turning the colony adeep-red color. To use TTC, flood the Petri plate with approximately 1mL of the 2% solution, and incubate the plate at 30°-35° C. forapproximately 2 hours. Microbial colonies will stand out sharply fromother material present on the plate and can be more easily counted.

C. Test for Total Combined Molds and Yeasts Count. 1. Prepare thesamples to be tested as described. To each plate, add 15-20 mL ofsterile SDA which has been melted and cooled to approximately 45° C.Cover each dish, and gently tilt or swirl the dish to mix the samplewith the agar and allow the contents to solidify at room temperature.Invert the plates and incubate for 5 to 7 days at 20°-25° C. [NOTE: Donot disturb the plates during incubation, because the dispersal of moldwithin the plate could yield a higher count than actually present.]

2. Following incubation, examine the plates for growth using amagnification device such as a Quebec Colony Counter, count the numberof colonies and calculate the results on a unit basis (per tablet,capsule, mL, gram, etc.) as designated in the materials specificationand evaluate for acceptability against the materials specification.

3. Further characterize fungal contamination by macroscopic andmicroscopic morphology. Where appropriate, subject yeasts to biochemicaltesting (or alternate suitable means of identification).

Procedure for Testing Absence of Objectionable Organisms

a) Sample Preparation—Unless otherwise directed by the Specific Method,prepare the sample for testing as described above in Sample Preparationsection for the Total Aerobic and Total Combined Molds and Yeasts. Referto the appropriate section below for additional information depending onwhich test is being performed.

b) Membrane Filtration—As an alternative to preenrichment procedures, asuitable, validated membrane filtration test procedure may be used. Thismay be especially useful for products containing inhibitory substances.

c) Retesting—For the purpose of confirming a doubtful result by any ofthe following procedures, a retest may be performed using two andone-half times (minimum) the initial sample size, with appropriatediluent adjustments.

B. Test for Absence of Staphylococcus aureus and Pseudomonas aeruginosa.To the sample add TSB to make 100 mL, mix and incubate for 24-48 hoursat 30°-35° C. Examine the medium for growth, and if present, using aninoculating loop, streak a portion to a selective medium, incubate andexamine for the characteristics listed below (commercially availableidentification kits may be substituted for the individual reactiontests):

Characteristics of Staphylococcus Aureus:

Medium: Vogel-Johnson Mannitol-Salt Baird-Parker Morphology: blackcolonies yellow colonies black, shiny surrounded by with yellow coloniesyellow zones zones surrounded by clear zones 2-5 mm Coagulase Test*:Positive positive positive Gram Stain: positive cocci positive coccipositive cocci (clusters) (clusters) (clusters *Transfer representativesuspect colonies from the selective agar to individual tubes containing0.5 mL mammalian (preferably rabbit or horse) plasma, incubate at 37°C., examine at 3-4 hours and subsequently at suitable intervals up to 24hours for coagulation.Characteristics of Pseudomonas aeruginosa

Medium: Centrimide: PSF* PSP* Morphology: Greenish colorless toyellowish greenish Fluorescence in UV Greenish yellowish blue light:Oxidase**: Positive positive positive Gram Stain: negative rods negativerods negative rods *For the Pigment test, streak representative suspectcolonies from the Centrimide Agar to PSF dishes and PSP dishes. Coverand incubate at 35° ± 2° C. for a minimum three days. Examine the platesunder UV light. **For the Oxidase test, confirm any suspect colonies bytransferring to strips or dishes impregnated withN,N-dimethyl-p-phenylenediamine dihydrochloride; if there is nodevelopment of a pink color changing to purple, the sample meets therequirements for the absence of Pseudomonas aeruginosa. Note thatcommercially available test kits which have been demonstrated to performin an acceptable fashion may also be used.

If no growth is observed, or if none of the colonies found conform tothe set of characteristics listed in the tables above, the sample meetsthe requirements of the test for absence of that organism.

Test for Absence of Salmonella Species and Escherichia coli

Transfer the sample to a sterile container to contain a total 100 mL ofFluid Lactose Medium and incubate at 30°-35° C. for 24-48 hours. Gentlyshake and examine for growth. (Comercially available identification kitsmay be substituted for the individual reaction tests.) a) Salmonella—Ifgrowth is present in the Fluid Lactose Medium:

1. Transfer 1.0 mL portions to 10 mL tubes of Fluid Selenite-CystineMedium and Fluid Tetrathionate Medium, mix and incubate at 30°-35° C.for 24-48 hours.

2. By means of an inoculating loop, streak portions from both media ontoBrilliant Green Agar Medium, Xylose-Lysine-Desoxycholate Agar Medium,and Bismuth Sulfite Agar Medium. Cover, invert, and incubate at 30°-35°C. for 24-48 hours and examine for the morphological characteristicslisted below:

Characteristics of Salmonella

Medium: Brilliant Green Xylose-Lysine- Bismuth Sulfite DesoxycholateMorphology: small, transparent, red, with or without black or greencolorless or pink to black centers white opaque (frequently surroundedby pink to red zone)

3. Further identification may be conducted by transferring suspectcolonies to a buttslant tube of Triple-sugar-Iron-Agar Medium by firststreaking the surface of the slant and then stabbing the wire wellbeneath the surface. Incubate at 30°-35° C. for 24-48 hours and examine.If no tubes show evidence of alkaline (red) slants and acid(yellow)butts (with or without concomitant blackening of the butt), the samplemeets the requirements of the test for the absence of the genusSalmonella.

b) Escherichia coli-If growth is present in the Fluid Lactose Medium: 1.By means of an inoculating loop, streak a portion to MacConkey AgarMedium. Cover, invert, and incubate at 30°-35° C. for 24-48 hours.

2. If none of the resultant colonies displays a brick-red appearance(with a possible surrounding zone of precipitated bile) and are gramnegative rods (Cocco-Bacilli), the sample meets the requirements of thetest for the absence of Escherichia coli.

3. If colonies match this description, transfer them toLevine-Eosin-Methylene Blue Agar Medium. Cover the dishes, invert andincubate at 30°-35° C. for 24-48 hours. If none of the colonies exhibitsboth a characteristic metallic sheen under reflected light and ablue-black appearance under transmitted light, the sample meets therequirements of the test for the absence of Escherichia coli.

Media Formulae 1. Phosphate Buffer pH 7.2

a) Stock Soution: Monobasic Potassium Phosphate 34.0 g; Water (distilledor deionized) 1000 mL; Sodium Hydroxide TS 175 mL. Dissolve 34 grams ofMonobasic Potassium; Phosphate in about 500 mL of water contained in a1000-mL volumetric flask. Adjust to pH 7.1-7.3 by the addition of SodiumHydroxide TS (about 175 mL), add water to volume and mix. Sterilize andstore under refrigeration (2°-8° C.) until use.

b) Working Solution. For use, dilute the stock solution with water at aratio of 1 to 800 and sterilize.

2. TSA (Trypticase Soy Agar/Soybean-Casein Digest Agar). PancreaticDigest of Casein 15.0 g; Papaic Digest of Soybean Meal 5.0 g; SodiumChloride 5.0 g; Agar 15.0 g; Water 1000 mL; pH after sterilization:7.3±0.2.

3. TSB (Trypticase Soy Broth/Soybean-Casein Digest Broth). PancreaticDigest of Casein 17.0 g; Papaic Digest of Soybean Meal 5.0 g; SodiumChloride 5.0 g; Dibasic Potassium Phosphate 2.5 g; Dextrose 2.5 g; Water1000 mL; pH after sterilization: 7.3±0.2; 4. SDA (Sabouraud DextroseAgar); Dextrose 40 g; Mixture of equal parts of Peptic Digest of animaltissue and pancreatic digest of Casein 10.0 g; Agar 15.0 g; Water 1000mL; Mix and boil to effect solution; pH after sterilization: 5.6±0.2.

5. FLM (Fluid Lactose Medium). Beef Extract 3.0 g; Pancreatic Digest ofGelatin 5.0 g; Lactose 5.0 g; Water 1000 mL; Cool as quickly as possibleafter sterilization. pH after sterilization: 7.1±0.2.

Example 50 Isoelectric Focusing Gel Analysis of CTLA4-I₂

The purpose of this example is to determine the isoelectric point,number of isoforms, and micro heterogeneity of CTLA4-Ig.

Materials

-   IEF Calibration Kit (pH 3 to 10), (Amersham Pharmacia, Catalog No.    17-0471-01) or (pH 2.5 to 6.5), (Amersham Pharmacia, Catalog No.    17-0472-01).-   Ampholine PAGplate gel: pH 4.0 to 6.5, (Amersham Pharmacia, Catalog    No. 80-1124-81).-   IEF Sample Applicators, (Amersham Pharmacia, Catalog No.    80-1129-46).-   IEF Electrode Strips, (Amersham Pharmacia, Catalog No. 80-1104-40).-   Phosphoric Acid (85%), (EMD, Catalog No. PX0995-6).

Equipment

-   Multiphor II Electrophoresis System, (GE Healthcare, Catalog No.    18-1018-06).

Preparation of Reagents

-   Anode Buffer Solution (0.1 M Glutamic Acid in 0.5 M Phosphoric Acid)    (wicks soaked with this are placed on the (+) side of the slab).-   Example:-   3.4 mL 85% Phosphoric Acid.-   1.47 g±0.02 g Glutamic Acid.-   HPLC Grade water.-   Combine above reagents in a 100 mL graduated cylinder, bring to 100    mL with HPLC Grade water, cap and invert several times to mix.-   Store solution at 2-8° C. for up to six months.-   Cathode Buffer Solution (0.1 M β-Alanine) (wicks soaked with this    are placed on the (−) side of the slab).-   Example:-   0.9 g±0.02 g β-Alanine.-   HPLC Grade water.-   Combine above reagents in a 100 mL graduated cylinder, bring to 100    mL with HPLC Grade water, cap and invert several times to mix.-   Store solution at 2-8° C. for six months.-   Fixing Solution (3.5% 5-Sulfosalicylic Acid in 12% Trichloroacetic    acid).-   Example:-   240 g±5.0 g Trichloroacetic Acid.-   70 g±2.0 g 5-Sulfosalicylic Acid.-   2000 mL HPLC water.-   Combine above reagents to make up to 2000 mL volume.-   Store solution at room temperature for up to three months.-   Staining Solution:-   Mix the GelCode Blue Reagent solution immediately before use by    gently inverting and swirling the bottle several times.-   Note: It is important to mix stain reagent before pouring or    dispensing to ensure that a homogeneous sample of reagent is used.

Staining Control Preparation:

-   Reconstitute the Carbonic Anhydrase II with HPLC Grade water to make    1.0 mg/mL stock solution.-   10 μL of stock solution is combined with 90 μL of HPLC Grade water    to get a 0.10 μg/μL working solution.-   Load 10 μL of the 0.10 μg/μL solution on the gel (1.0 μg load).

Procedure

-   Sample Dilution-   Prepare a 20 μg/10 μL solution of sample and reference material in    HPLC water.

${\frac{{Desired}\mspace{14mu} {concentration}\mspace{14mu} \left( {2\mspace{14mu} {µg}\text{/}{\mu L}} \right)}{{Sample}\mspace{14mu} {concentration}\mspace{14mu} \left( {50\mspace{14mu} {µg}\text{/}{µL}} \right)} \times 200\mspace{14mu} {µL}} = {{µl}\mspace{14mu} {of}\mspace{14mu} {sample}\mspace{14mu} {added}\mspace{11mu} {to}\mspace{14mu} a\mspace{14mu} 200\mspace{11mu} {µl}\mspace{14mu} {final}\mspace{14mu} {volume}}$     Example:${\frac{2\mspace{14mu} {µg}\text{/}{µL}}{50\mspace{14mu} {µg}\text{/}{µL}} \times 200\mspace{14mu} {µL}} = {{0.04 \times 200\mspace{14mu} {µl}} = {8\mspace{14mu} {µl}\mspace{14mu} {sample}\mspace{14mu} \left( {{add}\mspace{14mu} 192\mspace{14mu} {µl}\mspace{14mu} H\; P\; L\; C\mspace{14mu} {water}} \right)}}$

Apparatus and Gel Preparation

Connect the Multiphore II electrophoresis unit's cooling plate to theThermostatic Circulator and set the temperature at 10±2° C.

Note: Allow circulator at least 20 minutes to reach the abovetemperature.

Remove the gel from the refrigerator. Carefully cut along the sides ofthe envelope making sure not to cut the gel/gel support.

Add approximately 1.0 mL: HPLC Grade water to one edge of the coolingplatform.

Place one edge of the gel/gel support into the water so the capillaryaction carries the water across the entire edge of the gel. Slowly,making sure air bubbles are not trapped, apply the gel across thecooling platform. Additional HPLC Grade water may be applied if needed.

-   Remove transparent film from the gel surface.

Soak one electrode with Anode Buffer Solution and place on the edge ofthe gel nearest the (+) markings of the cooling platform.

Soak one electrode in the Cathode Buffer Solution and place on the otherside of the gel, which is nearest the (−) markings on the cooling plate.

After the electrode strips have been applied, carefully cut the excessusing a new razor blade, so that the strips end at the edge of the gel,and not the gel support.

Apply sample application pieces on the side nearest the (−) markings onthe cooling platform, making sure there is good contact between thesample application pieces and the gel. Note: Make sure the IEF sampleapplicators do not touch the cathode buffer soaked strip, do not wick upcathode buffer, and are sufficiently separated to assure each sampleruns separately. The sample applicators should be firmly placed on theslab gel.

Place the electrode holder of the Multiphor II unit and align theelectrodes along the center of the electrode strips on the gel. Connectthe two electrodes from the electrode holder to the base unit and placethe safety lid in position. Using adhesive tape, cover the holes in thesafety lid to prevent the gel from drying. Connect the electrodes to thepower supply.

-   Prefocus the gel with the power supply set at the settings below,    until the voltage reaches ≥300 V.

TABLE 1 Power supply settings to prefocus IEF gels. Run ParametersSetting Voltage (variable parameter) 2000 volts maximum Current(constant parameter) 25 mAmps Power (constant parameter) 25 watts

Gel Loading

After the gel has been prefocused, turn off the power supply, remove thesafety lid, disconnect the electrodes and remove the electrode holderfrom the Multiphor II unit.

Load samples onto the sample applicators in a specific sequence. Forthis procedure, make sure the (−) cathode buffer strip side is closestto the analyst. Samples are loaded from right to left. First make two ormore applications of the IEF calibration standards (lanes 1 & 2), one ofthe reference material (lane 3), one of the test sample #1 (lane 4), oneof the staining control preparation (lane 5), one of test sample #2(lane 6), one of the reference material (lane 7) and one of the IEFcalibration standard (lane 8).

Add the third and fourth sample by applying one application of referencematerial (lane 9), one application of test sample #3 (lane 10), oneapplication of the staining control reparation (lane 11), oneapplication of test sample #4 (lane 12), one application of referencematerial (lane 13) and one application of IEF calibration standard (lane14). The fifth and sixth samples are applied by repeating the samepattern as samples 3 and 4, using lanes 15 to 20. Sample loading patternis shown in Table 2.

TABLE 2 Sample Dilution and Gel Loading Pattern Working ProteinConcentration Loading Load Lane Description (μg/μL) Volume (μL) (μg) 1IEF pI Marker* — 10 — 2 IEF pI Marker — 10 — 3 Reference Material 2.0 1020 4 Sample 1 2.0 10 20 5 Staining Control 0.10 10 1.0 6 Sample 2 2.0 1020 7 Reference Material 2.0 10 20 8 IEF pI Marker — 10 — 9 Referencematerial 2.0 10 20 10 Sample 3 2.0 10 20 11 Staining Control 0.10 10 1.012 Sample 4 2.0 10 20 13 Reference Material 2.0 10 20 14 IEF pI Marker —10 — 15 Reference Material 2.0 10 20 16 Sample 5 2.0 10 20 17 StainingControl 0.10 10 1.0 18 Sample 6 2.0 10 20 19 Reference Material 2.0 1020 20 IEF pI Marker — 10 —

Electrophoresis. When the gel is loaded, place the electrode holder ofthe Multiphor Hunit and align the electrodes along the center of theelectrode strips on the gel. Connect the two electrodes from theelectrode holder to the base unit and place the safety lid in position.Using adhesive tape, cover the holes in the safety lid to prevent thegel from drying. Connect the electrodes to the power supply. Setappropriate voltage, current and power settings and begin the run.

TABLE 3 Power supply settings to run the IEF gels. Run ParametersSetting Voltage (variable parameter) 2000 volts maximum Current(constant parameter) 25 mAmps Power (constant parameter) 25 watts Time(constant parameter) 2.5 hours

After the gel has been run, turn off the power supply, remove thesafetylid, disconnect the electrodes, and remove the electrode holderfrom the Multiphor II unit. Carefully remove the electrode strips andthe sample applicators from the gel.

-   -   Note: You can place the slab gel directly in the fixing solution        and let the electrode strips and application pieces float off        the gel.

Remove the gel/gel support from the cooling plate and place in a flatdish containing a sufficient volume of fixing solution (step 3.3) tokeep the gel wet. Cover with plastic wrap and put on an orbital platformand incubate 20-60 minutes. Record start and stop time on the IEFworksheet.

-   -   NOTE: IEF gels left in fixative too long sometimes delaminate.        This is avoided by limiting the fix time to approximately one        hour.

4.5 Staining the gel.

After fixing, rinse the gel 3×5 minutes with a sufficient volume of HPLCGrade water to keep the gel wet. Record start and stop times on the IEFworksheet.

After mixing the GelCode Blue Stain Reagent solution before using (step3.4), place the gel in a sufficient volume of the stain to keep the gelwet. Incubate 15-24 hours in a tightly sealed container, to preventreagent evaporation. Record start and stop times on the IEF worksheet.

Stained gels are destained by replacing Stain Solution with HPLC Gradewater. Rinse the gel at least three water changes over a 1-2 hourperiod. Record start and stop times on the IEF worksheet. Afterdestaining, the gel is ready for scanning.

-   -   Note: Additional washes may be required to reduce background        staining on the IEF gel.

System Suitability

Isoelectric focusing standards should be easily distinguished frombackground. Protein standards that migrate to pI values between 4.0 and6.5 are visible on the gel. Protein standards with pIs that are outsidethis range are not visible on the gel.. The pI markers at 3.50, 4.55,5.20, and 5.85 are identified and labeled on the gel image.

TABLE 4 Components of the IEF calibration standards. Protein Standard pITrypsinogen 9.30 Lentil Lectin basic band 8.65 Lentil Lectin middle band8.45 Lentil Lectin acidic band 8.15 Myoglobin basic band 7.35 Myoglobinacidic band 6.85 Human carbonic anhydrase B 6.55 Bovine carbonicanhydrase B 5.85 β-Lactoglobulin A 5.20 Soybean Trypsin Inhibitor 4.55Amylogucoside 3.50

A staining control of carbonic anhydrase II standard (pI 5.4) at a lowlevel of protein loading (1.0 μg) is used for visualization of gelstaining consistency. This band must be easily distinguished from thebackground by visual inspection of the scanned gel image.

The CTLA4-Ig reference material should be enumerated at 10-22 bandswithin the pI range of 4.3 to 5.6.

The cumulative percent intensity of the most prominent bands of CTLA4-Igreference material should be ≥90% within the pI range of 4.3 to 5.3.

For reference material, confirm by visual inspection that there arethree major bands focused between the pI markers 4.5-5.2 (see Figure forthe major bands pattern).

Gel Scanning and Analysis

After electrophoresis and staining, all the gels are scanned using adensitometer. The image files are stored on the computer local harddrive/network and archived via the local area network. The analysis ofscanned gel images is performed using ImageQuant TL software (v2003.03).The scanning and analysis parameters are listed in Table 5. Scans aregenerated and quantitated using departmental procedures.

TABLE 5 Gel Scanning and Analysis Parameters Setting Scan ParametersScan Pixel Size 100 Scan Digital Resolution 12 bits Band DetectionParameters Minimum Slope Initial 100 Noise Reduction Initial 10 %Maximum Peak Initial 0 Lane % width Set at 90%

Note: The above procedures provide basic steps for the analysis of gelimages. The scanparameters in table are defined. The band detectionparameters in table are suggested initial settings. Adjustment of theband detection parameters may be necessary to accurately identify bandsdue to physical property changes of gel (such as gel shrinkage afterstaining/destaining) and alteration of gel band shape and shifting.Manually correct any missed or misidentified bands.

Scan the gels using the scan parameters defined in Table. All analysisand assessments of the gel are made from the scanned image. Open a gelimage file (scanned raw data) from <1D Gel Analysis> in ImageQuantTL. Goto <Contrast> on toolbar and lower the <Image Histogram> parameter untilall bands are clearly visible. Select <Lane Creation> and choose<Manual> to set up <Number of Lanes> to be analyzed. Adjust <Lane %Width> up to 100% to cover the gel lanes. Properly align single lanes ifnecessary. Use <Rolling Ball> method to subtract background. Detectbands using the initial <Minimum Slope>, <Noise Reduction>, <%MaximumPeak> settings listed in Table 3. Adjustment of these values isnecessary to accurately identify bands. Compute band pI value by usingthe standard pI marker from the labeled markers listed in the SystemSuitability Section for the pH/pI 4.0-6.5 gel. Skip the calibration andnormalization steps. Enumerate the number of bands within the 4.3 to 5.6pI range. Export the results into Excel sheet for further documentationand reporting. Calculation of Cumulative Percent Intensity of SamplesRelative to Reference Material. Note: The cumulative percent intensityfor a sample is the percentage of bands that migrate within the pI rangeof 4.3-5.3, as compared to 100% of all bands present in the lane. Thefollowing equation should be used for the calculation of cumulativepercent intensity of samples relative to reference material:

${{Sample}\mspace{14mu} {Relative}\mspace{14mu} {Percent}\mspace{14mu} (\%)} = {\frac{{Sample}\mspace{14mu} \% \mspace{14mu} {Band}\mspace{14mu} {Intensity}\mspace{14mu} \left( {{pI}\mspace{14mu} 4.3\text{-}5.3} \right)}{{Reference}\mspace{11mu} \% \mspace{14mu} {Band}\mspace{14mu} {Intensity}\mspace{14mu} \left( {{pI}\mspace{14mu} 4.3\text{-}5.3} \right)} \times 100}$

Note: Refer to the gel loading pattern, the reference material next tothe sample should be used for the calculation. If the reference materialhas a cumulative value of 100%, and the sample has a 95% value, thesample relative percent is 95/100*100=95%. If the reference has acumulative value of 95%, and the sample has a 100% value, the samplerelative percent is 100/95*100=105%.

Reporting Results

Report the number of bands enumerated within pI of range 4.3-5.6. Reportthe cumulative percent intensity relative to that of CTLA4-Ig referencematerial within the pI range of 4.3-5.3 (see Figure for an example ofthe report for the quantitative IEF gel analysis defined in thismethod). Confirm by visual inspection that there are three major bandsfocused between the pI markers 4.5-5.2 relative to reference material(see FIG. 1 for the major bands pattern) and report number of majorbands observed within pI markers 4.5-5.2. Confirm by visual inspectionthat there are no new significant bands between pI makers 4.5-5.2relative to reference material.

In certain embodiments, the results of this method will show bands in apI range of from 4.3-5.6, or 4.3-5.3, with there being identified from10 to 22 bands, with the cumulative band intensity from 90-110%. Inanother embodiment, the pI range is from 4.5-5.2 with 3 major bands. Inanother embodiment, the pI range is 4.3-5.6 with 10 to 22 bands. Inanother embodiment, the pI range is 4.3-5.3 with a cumulative bandintensity of from 95-105%. In another embodiment, the pI range is from4.5-5.2 with 2 prominent bands.

Example 51 SDS-PAGE of CTLA4-Ig

The example shows the examination of CTLA4-Ig under both reduced andnon-reduced conditions by SDS-PAGE.

Materials

-   Tris-Glysine (Tris-Gly) SDS sample buffer 2× (Invitrogen, Catalog    No.LC2676).-   NuPAGE “Sample Reducing Agent 10× (Invitrogen, Catalog No. NP0004).-   4-20% Tris-Glycine Gel—1.0 mm×12 wells (Invitrogen, Catalog No.    EC60252BOX).-   Tris-Glycine SDS Running Buffer 10× (Invitrogen, Catalog No.    LC2675).-   Mark12™ Wide-Range Unstained Standard (Invitrogen, Catalog No.    LC5677).-   GelCode Blue Stain Reagent (Coomassie Blue), (Pierce, Catalog No.    24590: 500 mL Catalog No. 24592: 3.5 L).-   SilverSnap® Stain Kit II (silver stain) (Pierce, Catalog No. 24612).-   Instrumentation:-   XcelI SureLock Mini Cell (Invitrogen, Catalog No. EI0001).-   Power supply for electrophoresis (OWL Separation Systems, Catalog    No. OSP-300).-   Reagents:-   Fixing solution for Coomassie Blue Stain (50% Methanol and 7% Acetic    Acid in HPLC Grade water).-   Example:-   To an appropriately sized, graduated container containing a stir    bar:-   Add 500 mL of Methanol.-   Add 70 mL Acetic Acid.-   Adjust volume to 1000 mL with HPLC Grade water.-   Store at room temperature for up to six months.-   Coomassie Blue Stain (GelCode Blue).-   Use straight from container. Add sufficient amount to cover the gel,    approximately 50 mL for one mini gel (10×10 cm) in a small tray.-   Store at 2-8° C. for up to one year.-   Silver Stain Fixing Solution (30% Ethanol and 10% Acetic Acid in    HPLC Grade water).

To an appropriately sized, graduated container containing a stir bar:Add 300 mL Ethanol. Add 100 mL Acetic Acid. Adjust volume to 1000 mLwith HPLC Grade water. Mix solution and store solution at roomtemperature for up to six months. Gel Wash Solution (10% Ethanol).

To a 50 mL centrifuge tube: Add 1.0 mL Enhancer (Silver Stain Kit). Add50 mL Silver Stain (Silver Stain Kit). Cap the tube and mix by gentlyvortexing for 3 to 5 seconds. Developer Working Solution. Prepareimmediately before use.

To a 50 mL centrifuge tube: Add 1 mL Enhancer (Silver Stain Kit). Add 50mL Developer (Silver Stain Kit). Cap the tube and mix by gentlyvortexing for 3 to 5 seconds. 1× Tris-Glycine SDS Running Buffer.Prepare on the day of use. To a graduated cylinder: Add 900 mL HPLCGrade water. Add 100 mL Tris-Glycine-SDS 10× Running Buffer. Combine theabove reagents cover with parafilm and invert several times to mix.

Staining Control. Add 1 mL of HPLC Grade water to a vial containing 2 mgTrypsin inhibitor (staining control). This will yield a 2 μg/μL stocksolution stable for six months at −200 C. To prevent degradation due tonumerous freeze thaw cycles, transfer 50 μL aliquots into small tubesand store at −200 C. Add 25 μL of stock solution to 75 μL of HPLC Gradewater to give a 0.5 μg/μL solution. Add 40 μL of the 0.5 μg/μL solutionto 160 μL of HPLC Grade water to give a concentration of 0.1 μg/μL. Add10 μL of the 0.1 μg/μL solution, 50 μL of 2× TrisGly Sample Buffer, and40 μL of HPLC Grade water to a microcentrifuge tube. The finalconcentration of the Trypsin inhibitor staining control is 0.01 μg/μL.Samples analyzed for release and stained with Coomassie Blue are loadedat a concentration of 10 mg per 10 μL.

For Coomassie Blue gels, dilute the Reference Material and sample inHPLC Grade water to a concentration of 10 μg/μL. Example: Add 80 μL of50 μg/μL Reference Material sample solution +320 μL of HPLC Grade water.For Non-reduced Samples, add 10 μL of the 10 μg/μL solutionconcentration to 50 μL of 2× Tris-Gly sample buffer, and 40 μL of HPLCGrade water into a microcentrifuge tube. For the Reduced Samples, add 10μL of the 10 μg/μL solution, to 50 μL of 2× TrisGly Sample Buffer, 30 μLof HPLC Grade water and 10 μL of 10× Reducing Agent. For Silver StainedGels, further dilute the 10 μg/μL solution to 1 μg/μL in HPLC Gradewater. Example: Add 40 μL of 10 μg/μL solution +360 μL HPLC Grade water.For Non-reduced Samples add 10 μL of the 1 μg/μL solution to 50 L of 2×TrisGly Sample Buffer, and 40 μL of HPLC Grade water in amicrocentrifuge tube. For the Reduced Samples, add 10 μL of the 1 μg/μLsolution, 50 μL of 2× TrisGly Sample, 30 μL of HPLC Grade water and 10μL of 10× Reducing Agent to a microcentrifuge tube. For the Blank,combine 50 μL of 2× TrisGly Sample Buffer and 50 μL of HPLC Grade waterin a microcentrifuge tube.

TABLE 6 The Molecular Weight (kDa) range for expected minor proteinbands that may be present in reduced and non-reduced abatacept samplesBand(s) description Non-Reduced (kDa) Reduced (kDa) Minor Band(s) NA15-45 Minor Band(s) 30-70 NA Minor Band(s) NA  80-155 Minor Band(s)175-230 175-200

Remove gel(s) from their wrapping and rinse the outside with HPLC Gradewater to remove polyacrylamide pieces. Carefully remove the well combmaking sure that the wells are straighted with a gel loading tip ifnecessary. Fill wells with HPLC Grade water and flick so that the wateris removed from the wells. Repeat well rinsing two more times. 5.2Insert gels into the XCell apparatus so that the short plate face facesthe inner chamber. If only one gel is being used, insert a plexiglassplate on the opposite side. Wedge the gel(s) tightly forming an innerand outer chamber. Fill the inner chamber with 1× Tris-Glycine SDSRunning Buffer. Check for leaks, then fill the outer chamber with 1×Tris-Glycine SDS Running Buffer. All gels must contain at least oneBlank lane and one Molecular Weight Marker lane. Add a Staining Controlfor Coomassie Blue stained gels only. Use gel loading tips. Load atleast one “Blank” between reduced and non-reduced samples. Treat theMolecular Weight Markers as reduced sample. This will help preventreduction of the non-reduced samples due to leaching of reducing agent.Attach the top of the Xcell apparatus and connect the electrodes to thepower supply. Adjust the current to 25 mAmps per gel, and set thevoltage (v) and power (w) to maximum. If running two gels, set thecurrent to 50 mAmps. Adjust the current when running two gels in oneapparatus, or multiple Xcell apparati on the same power supply.Electrophorese for 60±5 minutes or until the buffer front moves at least80% of the available migration distance. Record start time and stop timeon the worksheet. Carefully separate the two plastic plates holding thegel by prying with a gel knife. After separation of the gel, followprocedure for staining.

Coomassie Blue Staining—Place the gel in 50 mL of Coomassie Blue FixingSolution (50% Methanol and 7% Acetic Acid) for 15 minutes. Rinse the gel3 times with ˜100 mL of HPLC Grade water for 5 minutes, for a total of15 minutes. Add the Coomassie Blue stain directly to the gel(s) andincubate for 15 to 24 hours. Stained gels are destained by replacing theCoomassie Blue Stain reagent with 100 mL of HPLC Grade water. After 1hour of destaining, the gel is ready for scanning.

Gel Scanning and Analysis. After electrophoresis and staining, the gelsare scanned using a densitometer. The image files are stored on thecomputer local hard drive and/or network and archived via the local areanetwork. The analysis of scanned gel images is performed usingImageQuant TL software (v2003.03). Scans are generated and quantitatedusing departmental procedures.

TABLE 4 Gel Scanning and Analysis Parameters Setting Scan ParametersScan Pixel Size 100 Scan Digital Resolution 12 bits Band DetectionParameters Minimum Slope Initial 100 Noise Reduction Initial 10 %Maximum Peak Initial 0 Lane % width Set at 90%

By visual inspection, the major band of reduced CTLA4-Ig must appear asa broad band that migrates to a position proximal to the 55,400 Damolecular weight marker (Glutamic dehydrogenase).

Example 52 Determination of Chinese Hamster Ovary (CHO) Host CellProtein Impurities in CTLA4^(A29YL104E)-Ig Drug Substance by ELISA

This example describes an enzyme-linked immunosorbent assay (ELISA) toquantitate contamination levels of CD-CHO1 residual host cell proteins(HCP) in test samples. A rabbit polyclonal anti-CD CHO1 HCP IgG is firstcoated on a microtiter plate. HCP reference standards, quality controlsand CTLA4^(A29YL104E)-Ig drug substance samples are incubated with thebound rabbit anti-CD CHO1 HCP IgG. After washing the microtiter plates,polyclonal rabbit anti-CD CHO1 HCP Biotin IgG antibody is added whichbinds to the HCP captured during the initial step. The microtiter platesare washed to remove any unbound polyclonal antibodies.Streptavidin-horseradish peroxidase is added and the microtiter plate isagain washed to remove any unbound conjugated antibodies. TMB chromogenis then added to yield a colorimetric reaction. The reaction isterminated with sulfuric acid and absorbance is measured at 450 nm in a96-well microplate reader. Color develops in proportion to the amount ofHCP captured. Sample concentrations are determined based on a standardcurve generated by plotting the absorbance versus HCP concentration inthe range from 4.11 ng/mL to 3000 ng/mL.

The Chinese Hamster Ovary (CHO) cell line (DG44) is used in theproduction of CTLA4^(A29YL104E)-Ig. For the production of CD-CHO1protein (HCP), DG44 cells were stably transfected with the recombinantvector pD16 and grown in CD-CHO1 medium supplemented with galactose andRecombulin. The polyclonal antibodies for this version of the ELISA weregenerated in New Zealand white rabbits immunized with a concentrate ofthe CD-CHO1 HCP material. Rabbit antibodies were affinity purified(Protein A), then dialyzed into phosphate buffered saline andconcentrated. Approximately 50 mg of the IgG fraction of rabbitanti-CD-CHO1 antibody was biotinylated using N-hydroxysulfosuccinimideester chemistry. The unmodified rabbit ant-CHO1 antibody is used to coat96-well microtiter plates. It captures CD-CHO1 HCP which are detected bythe biotinylated rabbit anti-CD-CHO1 antibodies. StreptavidinHorseradish Peroxidase conjugate binds to biotin and a colorimetricreaction with TMB chromogen is used to quantify CD-CHO1 HCP.

This method, shown in FIG. 98, is designed to quantitatively determineresidual levels of CD-CHO1 host cell proteins by ELISA for releasetesting of CTLA4^(A29YL104E)-Ig drug substance material.

Rabbit anti-CD-CHO1 antibody is biotinylated using a biotinylation kitwith Sulfosuccinimidyl-6 (biotinamido) hexanote as biotinylatiomreagent. The biotinylation reagent from PIERCE (product # 21335) withSulfo-NHS-LC-Biotin can be used. The antibody is labeled according tothe manufacturer's recommendations in the manual supplied with thebiotinylation reagent. After labeling and separation on the suppliedsize exclusion column, the biotin incorporation is determined and thesample is frozen in aliquots of 50 μL or smaller at or below −70° C. Thebiotin/IgG ratio of the final product should be between 2 and 4. Storeat or below −70° C.

Plate Coating. Prepare an 8 μg/mL solution of purified rabbitanti-CD-CHO1 HCP IgG in Carbonate Buffer to be used for coatingmicrotiter plates (12 mL of solution is required per microtiter plate).Add 100 μL of this solution to each well of an Immulon 4 microtiterplate using a calibrated multichannel pipettor. Cover the microtiterplate with parafilm and incubate at 4° C. for 18±2 hours.

Plate Washing and Blocking. Wash plate three times with Wash Bufferusing plate washer instrument. Add 300 μL SeaBlock to each well using acalibrated multichannel pipettor. Incubate the plate for 1 hour at22.5±5° C. Prepare concentrations of CD-CHO1 Protein Reference Standardsand Quality Control samples in 15 mL graduated sterile polypropylenetubes. Dilute Reference material and Quality Control samples usingTeknova Diluent (1.21). Standard concentration are prepared on the dayof the experiment at the concentrations listed in the example below:

Dilutions for Standard Curve Samples (Example for Daily Preparation)

Stock concentration of CD-CHO1 protein is 5.7 mg/mL

Dilution A: 26.3 μL (5.7 mg/mL) + 4.973 mL PTB Diluent = 30 μg/mlDilution B: 0.6 mL (30 μg/mL) + 5.4 mL Diluent 3000 ng/mL 2 mL (3000ng/mL) + 4 mL Diluent 1000 ng/mL 2 mL (1000 ng/mL) + 4 mL Diluent 333.3ng/mL 2 mL (333.3 ng/mL) + 4 mL Diluent 111.1 ng/mL 2 mL (111.1 ng/mL) +4 mL Diluent 37.0 ng/mL 2 mL (37.0 ng/mL) + 4 mL Diluent 12.3 ng/mL 2 mL(12.3 ng/mL) + 4 mL Diluent 4.11 ng/mL 0 + 4 mL Diluent 0 ng/mL

QC Concentration Analysis, Storage, and Expiration

Quality Control (QC) samples at three different target concentrations(25, 100, and 700 ng/mL) are prepared and used fresh on the day ofanalysis or prepared in larger amounts and stored frozen in aliquots ator below −70° C. Frozen aliquots are analyzed in three independentexperiments. The average result from the three experiments is reportedin a Certificate of Analysis (COA) for each of the three QC samples. Onthe day of the experiment, frozen QC samples are thawed and analyzed asdescribed below. After thawing each QC sample is vortexed at mediumspeed 2-4 seconds. Frozen QC samples expire 6 months after preparationdate. They are used at their nominal concentration reported on the COA.Freshly prepared QC expires 24 hours after preparation.

Dilutions for Quality Control (QC) Samples (example)

-   -   Dilution A: 26.3 μL (5.7 mg/mL)+4.973 mL Diluent=30 μg/mL    -   233 82 L (30 μg/mL)+9.767 mL Diluent=700 ng/mL (QC 1)    -   1.43 mL (700 ng/mL)+8.57 mL Diluent=100 ng/mL (QC 2)    -   2.5 mL (100 ng/mL)+7.5 mL Diluent=25 ng/mL (QC 3)

Sample Preparation. Dilute drug substance samples to approximately 12.5,6.25, and 3.125 mg/mL.

-   -   Dilution A: 400 μL sample (˜25 mg/mL)+400 μL Diluent=12.5 mg/mL    -   Dilution B: 400 μL (˜12.5 mg/mL)+400 μL Diluent=6.25 mg/mL    -   Dilution C: 400 μL (˜6.25 mg/mL)+400 μL Diluent=3.125 mg/mL

Plate Washing. Wash plates three times with Wash Buffer using platewasher. Add 100 μL per well of each standard concentration, samples andQC samples in triplicate to the blocked and washed plate. Each QCconcentration is added twice to a total of six wells per plate. Seeplate map in the Method Attachment for suggested placement. Incubate for1 hour at 22.5±5° C. Repeat step washing 5 times. Dilute rabbitanti-CD-CHO1 HCP Biotin to 2 μg/mL in Teknova Buffer. Vortex thesolution approximately 2-4 seconds at medium speed. Add 100 μL per well.Incubate for 1 hour at 22.5±5° C. Repeat step washing 5 times. DiluteStreptavidin-HRP (SA-HRP) appropriately in Teknova buffer (example: a1/20,000 dilution usually results in acceptable absorbance readings).Add 100 μL of SA-HRP dilution to each well and incubate at 22.5±5° C.for 1 hour. Remove the TMB chromogen from refrigerator and decant aminimum of 10 mL per plate into a suitable container. Place in a darklocation and allow to come to room temperature. Repeat step washing 5times. Add 100 μL of TMB chromogen to each well and incubate at 22.5±5°C. for approximately 2 minutes. Stop chromogen reaction by adding 100 μLof 1 N H₂SO₄ to each well. Add Stop Solution in the same order to platesand wells as the chromogen was added to ensure the same reaction timesof chromogen with the enzyme in each well. Measure absorbance at 450 nmwith a reference wavelength of 630 nm on an appropriate 96 well platereader.

DATA ANALYSIS. The Softmax program template “CHO1 ELISA template.ppr” isset up to generate mean values, standard deviations, % CVs, calculatedconcentrations, curve fit parameters, etc. Standard Curve. Referencestandard data are fitted to a standard curve using a four-parametercurve fit function:

$Y = {\underset{\_}{\left( {{\left( {A - D} \right)/\left( {1 + \left( {{X/C^{}}B} \right)} \right)} +} \right.}D}$

-   -   Where:    -   Y=absorbance value (A₄₅₀-A₆₃₀)    -   A=absorbance value corresponding to the minimum asymptote    -   D=absorbance value corresponding to the maximum asymptote    -   C=absorbance corresponding to one half the absolute difference        between the maximum and minimum asymptote values (inflection        point).    -   B=the slope at the inflection point of the curve    -   X=concentration of CD-CHO1 HCP

The Softmax program template “CHO1 ELISA template.ppr” determines thecorrelation coefficient (R²) of the regression line for the standardsusing the calculated mean.

EXEMPLARY VALUES. Determine if the results for Standards, QC, andsamples meet the exemplary values listed below. Exemplary values for theStandards. The correlation coefficient (R²) for the Standard Curve mustbe ≥0.99. The mean background for the zero ng/mL standard concentrationmust be ≤0.2 absorbance units. If two or more of the seven nominalconcentrations of the Standard Curve, excluding zero and concentrationsbelow the QL (12.3 ng/mL), do not meet conditions 6.1.4 and 6.1.5, theassay is considered invalid and must be repeated. The mean of thecalculated values (ng/mL) at each standard concentration used todetermine the Standard Curve, excluding zero and concentrations belowthe QL (12.3 ng/mL), must be within 20% of the target (nominal) value.The coefficient of variation (% CV) of the triplicate absorbance valuesat each Standard concentration, excluding zero and concentrations belowthe QL (12.3 ng/mL), must be less than 20%. To ensure that at least twocongruent data points are available for calculation; the standards,quality controls and samples are loaded in triplicate wells. Analyzeeach triplicate value separately. Drop the value that lies furthest fromthe target. Recalculate the curve and reanalyze the exemplary values.

-   -   Example:

Target Value (ng/mL) Actual Value (ng/mL) 25 12 24 26

The single value that is furthest from the target value of 25 ng/mL is12 ng/mL. By eliminating the 12 ng/mL value from the triplicate, themean of the remaining values meet all of the exemplary values. If it isshown that the mean of the remaining two values still do not meet theexemplary values, then the single point is eliminated and the curve isrecalculated.

-   -   Example:

Target Value (ng/mL) Actual Value (ng/mL) 25 5.0 5.5 10

The mean value is >20% from the target regardless of which value iseliminated, therefore, the single point is dropped from the curve andthe curve will be recalculated.

Exemplary values for QC Samples. A QC sample is a set of six wells atthe stated concentration. At least four of the six wells for a QC samplemust be within 20% of the nominal for the QC sample to be acceptable.All three QC samples must be acceptable. If these exemplary values arenot met, the assay must be repeated.

Exemplary values for Test Samples. The absorbance value for the testsample assayed must be less than the highest QC. If the value exceedsthat of the highest QC, the test sample must be diluted sufficiently soas to obtain a mean value between 700 and 12.3 ng/mL. At least one ofthe three sample dilutions (12.5, 6.25, and 3.125 mg/mL) must fallwithin the range of the standard curve for a reportable result, unlessthe absorbance for all dilutions are below the QL of the assay. In thatcase the test samples are reported as <QL. The mean of the triplicateabsorbance values of the sample dilutions that are greater than the QLand fall within the range of the assay should exhibit a CV of less than20%.

CALCULATION AND REPORTING RESULTS OF CD-CHO1 HCP CONCENTRATION INSAMPLES. Calculation of CD-CHO1 HCP concentration in samples. Multiplythe mean sample results by the appropriate dilution factor (i.e. 2, 4,and 8) to obtain the concentration of CD CHO1 HCP in the undilutedsample in ng/mL for each of the dilutions. Determine the mean forresults of those dilutions that fall within the range of the assay.Divide the result from by the reported CTLA4^(A29YL104E)-Ig proteinconcentration (mg/mL) to obtain the concentration of CD CHO1 HCP inng/mg of CTLA4^(A29YL104E)-Ig.

-   -   Example Calculation:

${{Mean}\mspace{14mu} {sample}\mspace{14mu} {result}\text{:}\mspace{14mu} \underset{\_}{235\mspace{14mu} {ng}\mspace{14mu} {CD}\text{-}{CHO}\; 1\mspace{14mu} H\; C\; {P/{mL}}}} = {9.4\mspace{14mu} {ng}\text{/}{mg}}$  Protein  Conc.:  25.0  mg/mL

-   -   NOTE: ng CD-CHO1 HCP/mg product (ng/mg) is equivalent to parts        per million (ppm).

Example 53 Determination of Protein a Levels in CTLA4^(A29YL104E)-Ig byELISA

This enzyme-linked immunosorbant assay (ELISA) quantitates contaminationlevels of Protein A in CTLA4^(A29YL104E)-Ig test samples. Rabbitanti-Protein A is first coated on a microtiter plate. Protein Areference standards, quality controls, recovery controls, andCTLA4^(A29YL104E)-Ig samples are incubated with the bound rabbitanti-Protein A IgG. After washing the microtiter plates, biotinylatedmonoclonal anti-rabbit Protein A IgG antibody is added which binds tothe Protein A captured during the initial step. The microtiter platesare washed to remove any unbound monoclonal antibodies.Streptavidin-horseradish peroxidase is then added after one hour ofincubation; the microtiter plate is again washed to remove any unboundconjugated antibodies. TMB chromogen is then added to yield acolorimetric reaction. The reaction is terminated with sulfuric acid andoptical densities are measured at 450 nm in a 96-well microplate reader.Color develops in proportion to the amount of Protein A captured. Sampleconcentrations are determined based on a standard curve generated byplotting the optical density versus Protein A concentration in the rangefrom 0.188 ng/mL to 12 ng/mL. A method outline is shown in FIG. 99.

Plate Coating with Capture Antibody. Prepare a 1 μg/mL solution ofrabbit anti-Protein A antibody in Coating Buffer and add 100 μL of thesolution to each well of an Costar microtiter plate. Incubate at 4° C.for 18±2 hours.

Plate Washing and Blocking. Wash plates three times with Wash solutionusing the plate washer. Add 200 μL of SUPERBLOCK™ to each well. Incubatethe microtiter plate for 60 minutes at ambient temperature. Preparationof Reference Standard Prepare Reference Standard by mixing theappropriate amount of Protein A Reference Material into the appropriatevolume of acetate buffer. Vortex solution at medium speed for 2-4seconds. Incubate Reference Standard, Quality Control, and RecoveryControl samples for 10 minutes at ambient temperature before addition tomicrotiter plate.

Example: Reference Material Protein A, stock concentration 2.3 mg/mL

1:200 10 μL (2.3 mg/mL)+19904 (Acetate Buffer)=11500 ng/mL

1:479 10 μL (11500 ng/mL)+47804 (Acetate Buffer)=24 ng/mL

2 mL (24 ng/mL)+2 mL (Acetate Buffer)=12 ng/mL

2 mL (12 ng/mL)+2 mL (Acetate Buffer)=6 ng/mL

2 mL (6 ng/mL)+2 mL (Acetate Buffer)=3 ng/mL

2 mL (3 ng/mL)+2 mL (Acetate Buffer)=1.5 ng/mL

2 mL (1.5 ng/mL)+2 mL (Acetate Buffer)=0.75 ng/mL

2 mL (0.75 ng/mL)+2 mL (Acetate Buffer)=0.375 ng/mL

2 mL (0.375 ng/mL)+2 mL (Acetate Buffer)=0.188 ng/mL

0 mL+2 mL (Acetate Buffer)=0 ng/mL

Each standard concentration is analyzed in triplicate. Place samples onmicrotiter plate as described in the method attachment. Preparation ofthe Quality Control Samples. Quality Control (QC) samples of Protein Aare prepared in acetate buffer at three target concentration levels of0.5, 2, and 5 ng/mL. They are either prepared fresh on the day of theexperiment or in larger quantities and frozen in 750 μL aliquots at≤−70° C. The Protein A concentrations in the frozen QC samples arepre-determined in three independent Protein A ELISA experiments usingthis method and the average concentration results are reported as the“nominal” QC concentrations in a Certificate of Analysis for each QCsample.

On the day of an experiment, thaw one vial each of the three QC samplesat room temperature. Analyze each QC concentration twice (in twotriplicate analyses per concentration). Place QC samples on microtiterplate as described in the method attachment.

Reference Material Protein A, 2.3 mg/mL

1:200 10 μL (2.3 mg/mL)+19904 (Acetate Buffer)=11500 ng/mL

1:479 10 μL (11500 ng/mL)+47804 (Acetate Buffer)=24 ng/mL

833.34 (24 ng/mL)+3.166 mL (Acetate Buffer)=5 ng/mL (QC1)

1.600 mL (5 ng/mL)+2.4 mL (Acetate Buffer)=2 ng/mL (QC2)

1 mL (2 ng/mL)+3 mL (Acetate Buffer)=0.5 ng/mL (QC3)

Preparation of the Test Samples. Prepare concentrations of 2.5 mg/mL,1.25 mg/mL, and 0.625 mg/mL of the CTLA4^(A29YL104E)-Ig test samples inpolypropylene tubes using acetate buffer (pH 3.5). Test samples areincubated for approximately 10 minutes at ambient temperature beforeadding to microtiter plate.

Plate Washing. Wash plates three times with wash solution using theplate washer. Plate washer should be set to fill wells with 300 μL washbuffer, zero soak time. Add 100 μL of each Reference Standard, QualityControl, Recovery Control, and test samples to each well and incubatefor one hour at ambient temperature. Repeat Step. Dilute biotinylatedanti-protein A antibody with Teknova diluent to a desired concentrationas indicated by optimization for each new lot. For example, make1:64,000 dilution for the monoclonal anti-protein A biotin conjugate,vortex at medium speed and add 100 μL to each well using a multichannelpipettor. Incubate at ambient temperature for one hour.

Dilute Streptavidin-Horseradish Peroxidase with Teknova diluent to adesired concentration as indicated by optimization for each new lot. Forexample, make 1:80,000 dilution for the Streptavidin-HorseradishPeroxidase. Vortex at medium speed and add 100 μL to each well andincubate for 30 minutes at ambient temperature. Repeat Step but, washfive times. Add 100 μL TMB chromogen to each well. Incubate at roomtemperature for approximately 2 minutes. The optical density for thehighest concentration for the standard curve should be between 0.980 and1.400. Stop chromogen reaction by adding 100 μL/well of 1 N H₂SO₄. Addstop solution in the same order to plates and wells as the chromogen wasadded to ensure the same reaction times of chromogen with the enzyme ineach well. Measure absorbance at 450 nm with a reference wavelength of630 nm on an appropriate 96 well plate reader.

DATA ANALYSIS. Refer to the Softmax program template for the Protein AELISA as it generates mean, standard deviations, and % CVs, etc. All ofthe calculations performed in Data Analysis sections will be performedusing the Protein A ELISA template.ppr in SoftMax Pro.

Average the triplicate absorbance values (Abs) obtained for eachreference and sample concentration assayed. Model the data for theProtein A standards using an unweighted four parameter regression of theform:

${Abs} = {\frac{\min - \max}{1 + {\left( {C/{ED}_{50}} \right)B}} + \max}$

-   -   Where:    -   Abs=absorbance    -   min=absorbance value corresponding to the minimal asymptote    -   max=absorbance value corresponding to the maximal asymptote    -   ED₅₀=absorbance corresponding to one half the absolute        difference between the maximal and minimal asymptotic values    -   B=the slope the inflection point of the curve fit    -   C=Concentration of Protein A

EXEMPLARY VALUES. Exemplary values for the Standards. The exemplaryvalues for the standards applies to those values at or above thequantitative limit (QL), as values below the QL are used only to helpestablish the extremes of the curve. The coefficient of determination(R²) for the standard curve should be ≥0.99. The mean background for thezero ng/mL standard should be ≤0.08 absorption units. The mean of thecalculated values (ng/mL) at each standard concentration used todetermine the standard curve except zero and the QL must be within 15%of the target (nominal) value. The mean of the triplicate absorbancevalues of the QL of the standard curve must exhibit a % CV of less than20% and be within 20% of target.

Example 54 ELISA for the Determination of MCP-1 like Protein inCTLA4^(A29YL104E)-Ig

This ELISA is performed to determine the concentration of MCP-1 likeprotein in CTLA4^(A29YL104E)-Ig. The concentrations of a standard curve(0.4 to 25.6 ng/mL), Quality Control, and test samples are applied togoat anti-mouse MCP-1 absorbed microtiter plates, and incubated for 60minutes at ambient temperature (22.5±5° C.). Plates are washed,secondary antibody (rabbit anti-rat MCP-1 IgG) is added, and incubatedfor 60 minutes at 22.5±5° C. Plates are washed, goat anti-rabbit IgGHorseradish Peroxidase is added to each well, and incubated for 30minutes at 22.5±5° C. Plates are washed and TMB Chromogen is added toyield a colorimetric reaction. After stopping the reaction with 1NH₂SO₄, optical densities are measured at 450 nm in a 96-well microplatereader, and the data is modeled using a 4-parameter regression curve.The concentration of MCP-1 like protein is then calculated for each testsample relative to the MCP-1 reference material. The concentration isreported in ng MCP-1 like protein per mg (parts per million, ppm) ofsample by dividing the result obtained relative to the standard curveand the undiluted sample concentration. This method allows for thedetermination of MCP-1 like impurities in cell culture derivedbiological samples including in-process, purified drug substance, anddrug product. MCP-1 like protein may be present in biological samplesproduced in Chinese Hamster Ovary (CHO) cell culture. Two polyclonalantibodies were identified that bind to an MCP-1 like impurity purifiedfrom CHO cells. The antibodies are directed against murine and rat MCP-1(intact) and both do cross react with MCP-1 like protein from CHO cellswhich is demonstrated in this report.

Plate Coating. For coating, dilute goat anti-mouse MCP-1 antibody to 5μg/mL with Coating Buffer. Coat plates with 100 μL/well. Cover plateswith plate sealers and incubate at 22.5±5° C. for 12 to 18 hours. PlateWashing. Wash plates three times with Wash Solution using a platewasher. Washer should be set at three washes, 300 μL/well with a zerosoak time. Alternatively, plates may be washed manually. Add 300 μL ofsolution to each well of each plate using a calibrated multichannelpipettor. Empty wells by flicking out into a sink and blot gently onpaper towels. Repeat three times.

Plate Blocking. Add 300 μL Coating Stabilizer & Block Buffer to eachwell using a calibrated multichannel pipettor. Incubate the plates forone to two hours at 22.5±5° C. Plate Washing and Storage. Wash platesthree times with Wash Solution as described under 4.2. Fill plates with300 μL Coating Buffer per well, cover with plate sealers and store inthe dark at 2-8° C. for up to one week.

Preparation of the Standards. From the MCP-1 like protein stock, preparea 25.6 ng/mL solution in PTB and dilute from there in serial dilutionsteps (1:2) to 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, and 0 ng/mL using PTB asdiluent. Alternatively, frozen standards can be used by preparing alarge quantity of standards. Aliquot and store at -70° C. to −80° C.Avoid repeated freeze/thaw. Frozen Standards need to be qualifiedagainst Quality Controls in three independent ELISA runs. The FrozenStandards and QC must be acceptable in each run. After completion ofthree acceptable runs, a Certificate of Analysis is issued. FrozenStandards will be used at their nominal concentrations. They expire 3months from the date of preparation.

Example for a MCP-1 Reference with a stock concentration of 0.97 mg/mL:

-   -   Prepare 2.5 mL of 5200 ng/mL in PTB:        -   13.4 mL stock+2.487 mL Diluent    -   b) Prepare 160 mL of 25.6 ng/mL in PTB:        -   788 mL stock a+160 mL Diluent        -   Mix each solution by vortexing the tube for approximately            2-3 seconds and then gently inverting 2-3 times.

Quality Control. Quality Controls (QCs) are MCP-1 like protein preparedat nominal concentrations of 17.7 ng/mL, 5.3 ng/mL, and 1.2 ng/mL inPTB. Prepare a large quantity of QCs, aliquot, and store at −70° C. to−80° C. for up to 3 months. Avoid repeated freeze/thaw. The QualityControls are qualified against standard curve in three independent ELISAruns. The QC must be acceptable in each run. After completion of threeacceptable runs the average result for each acceptable QC is reportedand a Certificate of Analysis is issued. QC will be used at theircalculated concentrations. They expire three months from the date ofpreparation. Example for preparing frozen QC dilutions from an MCP-1Reference with a stock concentration of 0.97 mg/mL:

-   -   a) Prepare 2.5 mL of 5200 ng/mL in PTB:        -   13.4 mL stock+2.487 mL Diluent    -   b) Prepare final dilution of MCP-1 like protein Quality Control        samples as follows:

5200 ng/mL MCP-1 Amount of Concentration of Like Protein to PTB BufferTotal MCP-1 (ng/mL) Add (μL) to Add (mL) Volume (mL) 17.7 409 119.6 1205.3 122 119.9 120 1.2 28 120 120

Mix each solution by vortexing 2-3 seconds and then gently inverting 2-3times. Alternatively, QC can be prepared fresh on the day of analysis.

Test samples. Test samples are prepared by adding 200 μL of the testsample to 200 μL of PTB and vortex for 2-4 seconds. Add referencestandards, quality controls (x2), and test samples to the plate, intriplicate, 100 μL per well and incubate for 1 hour at 22.5±5° C. (donot use outer wells). Prepare secondary rabbit anti-rat MCP-1 antibodyin PTB buffer at a concentration of 2 μg/mL in sufficient volume forplates used in the assay. Vortex the solution approximately 2-4 seconds.Repeat wash, but wash five times. Add 100 μL of the secondary antibodysolution to each well and incubate for 60 minutes at 22.5±5° C. Preparetertiary goat anti-rabbit HRP conjugate solution (eg. 1:20,000 dilutionor appropriate dilution) using PTB as diluent. Vortex the solutionapproximately 2-4 seconds. Repeat wash. Add 100 μL HRP conjugate to eachwell and incubate at 22.5±5° C. in the dark for 30 minutes. Repeat wash.Add 100 μL of TMB to each well and incubate at 22.5±5° C. in the darkfor 3-6 minutes or until appropriate color has developed. Stop chromogenreaction by adding 100 μL of 1N H₂SO₄ in the same order as the additionof chromogen was made. Read optical densities at 450 nm with a referencewavelength of 630 nm on an appropriate 96-well plate reader.

Data Reduction

-   -   Use the Softmax™ Software with the protocol file MCP-1.ppr to        construct a standard curve using an unweighted four parameter        regression of the form.

${Absorbency}_{450} = {\frac{A - D}{1 + \left( \frac{x}{c} \right)^{b}} + D}$

-   -   -   Where:        -   A=Absorbency₄₅₀ value corresponding to the minimal            asymptote.        -   D=Absorbency₄₅₀ value corresponding to the maximal            asymptote.        -   c=concentration corresponding to one half the absolute            difference between the maximal and minimal asymptotic            values.        -   B=the approximated slope of the linear portion of the curve.        -   x=concentration of reference standard.

Report results only for samples with acceptable data for standardcurves, quality controls, and test samples.

Exemplary values for the Standards. The exemplary values for thestandards apply to those values at or above the QL (0.8 ng/mL), asvalues below the QL are used only to help establish the extremes of thecurve. The mean background (the zero ng/mL standard) must be ≤0.1absorption units. The mean back-calculated MCP-1 concentration (ng/mL)at each standard concentration used to determine the standard curve,must be within ≤15% of the target (nominal) value. The coefficient ofvariance (% CV) of the triplicate A₄₅₀ values at each standardconcentration used to determine the standard curve, must be 15%. Themean of the triplicate A₄₅₀ values at the QL of the standard curve mustexhibit a % CV of ≤20% and back-calculate to within 20% of the target(nominal concentration, 0.8 ng/mL). Each value of the triplicate used tocalculate the mean will be analyzed separately. The value that liesfurthest from the mean will be dropped, the curve re-calculated, and theexemplary values re-analyzed. If it is shown that the mean of theremaining two values still do not meet the exemplary values, then thesingle point is eliminated and the curve is re-calculated.

Exemplary values for QC Samples. A QC sample is defined as a set ofthree wells at the stated concentration, therefore, for the threenominal concentrations stated in this method there are a total of six QCsamples. The back-calculated concentrations of at least two of the threewells for a QC sample must be within 20% of the previously determinedtarget concentration (see COA) for the QC sample to be acceptable. Atleast four of the six QC samples must be acceptable; two of the six QCsamples (not two at the same concentration) may be unacceptable and notmore than 6 of the 18 QC sample wells may deviate more than 20% from therespective target concentrations. If these exemplary values are not met,the assay must be repeated.

Exemplary values for Test Samples. The mean calculated MCP-1concentration must be less than the concentration of the Highest QC. Ifthe mean calculated MCP-1 concentration is 0.8 ng/mL (the QL) the % CVof the triplicate determinations must be 20%. If this condition is notmet the sample result is not valid and must be repeated. If thiscriterion is met, the average MCP-1 concentration is used to calculatethe final result. If the calculated MCP-1 concentration is <0.8 ng/mL(QL) the sample is reported as <QL and the value “<0.8 ng/mL” is used incalculation of the final result.

Example 55 Detection of Cho DNA in CTLA4^(A29YL104E)-Ig and CTLA4-Ig byQuantitative Polymerase Chain Reaction

This procedure was developed to detect residual CHO DNA in samples ofdrug substance using a real-time quantitative polymerase chain reaction(qPCR) assay. PCR is the replication of a mixture of DNA. This assayuses a fluorogenic probe to detect a specific CHO PCR product as itaccumulates during the assay. The rate of amplification of the PCRproduct is directly proportional to the amount of starting DNA presentin the sample.

Conversion of DNA Value to picogram/milligram. The pg/mL value isdivided by the concentration of the sample, determined by the absorbanceat 280 nm. Example calculation for a sample having a proteinconcentration of 50 mg/mL and a reported value from Bioreliance=0.67fg/μL. Step 1: Conversion to pg/mL=0.67 pg/mL. Step 2: Conversion topg/mg=(0.67 pg/mL/50.0 mg/mL)=0.013 pg/mg.

Example 56 Analysis of CTLA4^(A29YL104E)-Ig by SDS-Page

This example describes the analysis of CTLA4^(A29YL104E)-Ig for theassessment of purity for CTLA4^(A29YL104E)-Ig drug substance and drugproduct. CTLA4^(A29YL104E)-Ig is an engineered fusion protein consistingof a modified ligand binding domain of cytotoxic T lymphocyte antigen 4(CTLA4) and the Fcyl region of human IgG. CTLA4^(A29YL104E)-Ig has amolecular weight of ˜92 kDaltons and an apparent molecular weight of 97kDalton (non-reduced) or 55 kDalton (reduced). Electrophoresis ofCTLA4^(A29YL104E)-Ig on 4-20% gradient SDS-polyacrylamide gels separatethe main monomeric species from higher molecular weight species(aggregates, dimers, and higher order multimers) as well as any lowmolecular weight species (degradation fragments). Densitometric scanningand ImageQuantTL quantitation of Coomassie Blue stained gels yield ameasure of protein purity. Results are reported as percent purity ofCTLA4^(A29YL104E)-Ig under reduced and non-reduced conditions.

Reagents

NOTE: All reagents may be substituted with equivalent alternatives. 1×Tris-Glycine-SDS Running Buffer. Add 100 mL Tris-Glycine-SDS 10× Runningbuffer to a 1 liter graduated cylinder. Q.S. to 1 liter with Milli-Q orHPLC grade water. Cover, invert several times to mix. This reagentshould be prepared on the day of assay. NOTE: Prepare additional volumeif needed.

Coomassie Blue Staining Reagent. Mix the GelCode Blue Stain Reagentsolution immediately before use by gently inverting or tipping andswirling the bottle several times. Such mixing is especially importantwhen using the 3.5 L GelCode container with a manual pump (Catalog No.24592). Do not shake bottle to mix the solution. NOTE: It is importantto mix the stain reagent before dispensing to ensure that the solutionis homogeneous.

Fixing Solution: 50% Methanol/7% Acetic Acid in Water. Combine 500 mLmethanol and 70 mL acetic acid in a 1 liter graduated cylinder. Q.S.with Milli-Q water to 1 liter and mix. The fixing solution is stable atroom temperature for up to six months. NOTE: The fixing solution shouldbe prepared in a chemical fume hood.

Staining Control Preparation. Reconstitute the trypsin inhibitor to makea 2 mg/mL stock solution using Milli-Q water. Prepare 50 μL aliquots andfreeze at −30±10° C. for up to 6 months. Use the following dilution planto dilute the stock protein solution to a working concentration:

25 μL of stock solution+75 μL Milli-Q water=0.5 μg/μL

40 μL of 0.5 μg/μL+160 μL Milli-Q water=100 ng/μL

Use Table directly below to prepare the Staining Control loadingsolution.

Sample Preparation for Staining Control Stain Control PreparationNon-Reduced (μL) Trypsin Inhibitor (100 ng/μL) 10 Tris-Glycine SDSSample Buffer (2X) 50 Milli-Q Water 40 Total Volume 100

-   -   Loading 10 μL of the Staining Control Preparation will yield a        protein load of 100 ng.

Standard and Sample Preparation

Loading Pattern for Fixed Process, GLP/GMP Drug Substance, Drug Product,or Stability Samples. Dilution for Coomassie Blue Stained Gels. Dilutetest articles and reference material to the working concentration of 1.0μg/μL and load both reduced and non-reduced with molecular weightmarkers as described in Table directly below.

Sample Dilution and Gel Loading for Coomassie Blue Stained Gel WorkingLoading Protein (NR/R) Concentration Volume Load Lane DescriptionCondition (μg/μL) (μL) (μg) 1 Sample 1 NR 1.0 10 10 2 Reference NR 1.010 10 Material 3 Blank¹ NR 0.0 10 0 4 Sample 1 R 1.0 10 10 5 Reference R1.0 10 10 Material 6 Molecular Wt. R — 10 — Stds. 7 Reference R 1.0 1010 Material 8 Sample 2 R 1.0 10 10 9 Blank¹ NR 0.0 10 0 10 Reference NR1.0 10 10 Material 11 Sample 2 NR 1.0 10 10 12 Staining Control NR 0.0110 0.1 ¹Blank = Load 10 μL of 1X NR sample buffer NOTE: The stainingcontrol band is included on the scanned gel image for visual inspectionto assess system suitability. The staining control band is absent in thecropped gel image to maintain consistency of gel reporting.

Sample Preparation and Electrophoresis. Sample Dilution for a 10 μg/LaneProtein Load. Follow the method below to prepare samples for a 10 μg/10μL load. Using Milli-Q water as the diluent, dilute test samples andreference material to 10 mg/mL CTLA4A29YL104E-Ig. Approximateconcentrations may be used for calculations. For example: If a sample ofCTLA4A29YL104E-Ig drug substance has a concentration of 25 mg/mL,prepare a 1:2.5 dilution (add 40 μL of sample to 60 μL of Milli-Qwater). Follow Table directly below to prepare the final sample forelectrophoresis. Use microfuge tubes for these dilutions.

Dilution of Test Samples Non-Reduced Reagent Reduced (μL) (μL) TestArticle at 10 mg/mL 10 10 Tris-Glycine SDS Sample Buffer (2X) 50 50NuPAGE Reducing Agent (10X) 10 NA Milli-Q Water 30 40 Total Volume 100100

NOTE: Adjust the volume of protein solution and Milli-Q water as neededto achieve a final volume of 100 μL. NOTE: If the sample concentrationis <10 mg/mL, prepare the final sample according to the Table above.Adjust the volume of protein solution and water to maximize the proteinload on the gel.

Sample Heating. After preparing sample dilutions, close the microfugetubes and vortex the tubes to mix the solution. Heat sample(s) in awater bath at 80±5° C. for 2.0±0.5 minutes (use calibrated timer).Remove sample (s) from the heat and allow them to cool to roomtemperature. Invert tubes several times to remove condensation from thetop and sides of the tubes.

Apparatus and Gel Preparation. Remove gel from its packaging andcarefully remove the comb making sure that the walls of the wells arestraight. The wells can be straightened with a gel loading tip ifnecessary. Insert the gel into the electrophoresis unit so that theshort glass plate faces the inner chamber. If only a single gel is to berun, insert a plexiglass spacer on the opposite side. Wedge the gel(s)tightly to seal the inner chamber from the outer chamber. Completelyfill the inner chamber with 1× Tris-Glycine-SDS running buffer. Checkfor leaks, then fill outer chamber to the bottom of the wells with 1×Tris-Glycine SDS running buffer. Gently rinse wells using a pipette with1× Tris-Glycine-SDS running buffer to remove any residual acrylamide.Repeat well rinsing until wells are completely clear and defined.

Sample Loading . Using gel loading tips, load each well with 10 μL ofsample. Fill all blank lanes with 10 μL of 1× Non-Reducing SampleBuffer. This will help prevent reduction of the non-reduced sample dueto leaching of the reducing agent and will maintain a similar saltconcentration across the entire gel.

Electrophoresis. Attach the gel box cover, and connect the electrodes tothe power supply. Adjust the current to 25 mAmps/gel (mA) and set thevoltage (v) and power (w) to maximum. NOTE: Power supply setting mayvary from vendor to vendor. Adjust setting to achieve 25 mA/gel.Electrophorese the gel for 60 minutes or until the sample buffer dyefront just reaches the bottom of the gel. Turn off the power supply,disconnect leads, and remove gel(s) from device. Carefully pry theplastic plates apart. Hold the plastic plate with the gel attached overthe appropriate fixing solution for the staining technique. Submerge gelinto the solution until the gel dislodges from the plastic plate.

Gel Fixing. NOTE: All steps are performed at room temperature withgentle rocking on the orbital shaker. NOTE: Perform staining in atightly sealed container to prevent reagent evaporation. NOTE: Althoughthe volume cited can be used, it is imperative that the gel becompletely covered in all steps. The size of the gel and staining traymust be taken into account in determining volumes needed. Afterelectrophoresis, add 50 mL of the fixing solution (50% methanol/ 7%acetic acid solution) for 15 minutes. Rinse the gel 3 times for 5minutes each with ˜100 mL Milli-Q water. Mix the Coomassie Stain Reagentsolution before use, and add 50 mL for an 8×10 cm mini gel. Additionalreagent may be required if a larger tray is used. Gently shake the trayusing an Orbital Shaker for 20±1 hours. For consistency, stain all gelsin the same run for the same duration. Destain by replacing theCoomassie Stain Reagent with 100 mL of Milli-Q water. After 1 hour ofdestaining, the gel is ready for scanning.

Gel Scanning and Analysis

After electrophoresis and staining, all gels are scanned using adensitometer and analyzed using ImageQuantTL™ software. The image filesare stored on the computer local hard drive and archived via the localarea network. The scanning and analysis parameters are listed in theTable directly below.

Gel Scanning and Analysis Parameters Setting Scan Parameters Scan PixelSize 100 Scan Digital Resolution 12 bits Band Detection ParametersBackground Correction Radius set at 200 Minimum Slope Initial 500 NoiseReduction Initial 5 % Maximum Peak Initial 0 Lane % width Set at 75%NOTE: The Scan Parameters in this Table must not be changed duringscanning. The Band Detection Parameters, Lane % width (set at 75%) andBackground Correction (set at 200 Radius), are recommended for allscanned gel image analysis (any changes will need to be documented).Adjustment of Minimum Slope, Noise Reduction, and % Maximum Peakparameters may be necessary to accurately identify bands due todifferences in the physical properties of the gel, such as gel shrinkageafter staining/destaining or differences in the shape of the gel bandshape. Manually correct any missed or misidentified bands. Refer to theImageQuantTL (v2003.03) manual and on-screen instructions for additionalinformation for band detection parameters.

Scan the gel using the scan parameters listed in above Table. Allanalysis and assessments of the gel should be made from the scannedimage. Open a gel image file (scanned image) from <1D Gel Analysis> inthe ImageQuantTL software. Go to <Contrast> on toolbar and set the<Image Histogram-High> parameter to 0.3 to enhance the gel image forclear visualization of all bands. NOTE: This step is for easyvisualization of the gel image for the following analysis and has noimpact on the quantitative result. Do not use the enhanced gel image forthe purpose of visual assessment of gel bands or gel reporting. Select<Lane Creation> and choose <Manual> to set up <Number of Lanes> to beanalyzed. Set <Lane % Width> to 75%. Properly align single lanes ifnecessary. Use the <Rolling Ball> method to subtract the background. Toaccurately account for background, set <Radius> to 200. Detect bandsusing the initial <Minimum Slope>, <Noise Reduction>, and <% MaximumPeak> settings listed in Table 4. Adjustment of these values may benecessary to accurately identify bands. Manually assess any missed ormisidentified bands. Determine the band molecular weight by using themolecular weight marker listed below. Skip the calibration andnormalization steps. Export the results including the molecular weights,band volume, and band % into an Excel worksheet for furtherdocumentation and reporting. Eleven of the twelve molecular weightstandards listed in Table 5 should be easily distinguished frombackground (FIG. 78). Note: The Insulin B chain (3,500 Da) and Insulin Achain (2,500 Da) may appear as a single broad band or the Insulin Achain may not be visually identified on the gel.

Mark 12 Molecular Weight Standards Molecular Weight Protein Marker(Daltons) Myosin (rabbit muscle) 200,000 β-galactosidase (E. coli)116,300 Phosporylase B (rabbit muscle) 97,400 Bovine serum albumin66,300 Glutamic dehydrogenase (bovine liver) 55,400 Lactatedehydrogenase (porcine muscle) 36,500 Carbonic anhydrase (bovineerythrocyte) 31,000 Trypsin inhibitor (soybean) 21,500 Lysozyme (chickenegg white) 14,400 Aprotinin (bovine lung) 6,000 Insulin B chain (bovinepancreas) 3,500 Insulin A chain (bovine pancreas) 2,500

In this example, the major bands of the test article, for bothnon-reduced (monomer) and reduced (single chain), are to be in the samerelative position on the gel as the CTLA4A29YL104E-Ig referencematerial. See FIG. 78. The staining control of soybean trypsin inhibitor(21,500 Da) standard at 100 ng/load must be visible on the scanned gelimage (FIG. 78, lane 12). With the exception of a minor band undernon-reducing condition that is commonly observed (single chain) proximalto the 55,400 Da molecular weight marker, the relative percent intensityof any additional band in the Coomassie blue stained gel should be ≤2%for reference material. NOTE: A molecular weight estimation of the mainband cannot be accurately determined due to its non-gaussiandistribution. Visual inspection of the reduced reference material is toyield a single broad band migrating to a position proximal to the 55,400Da molecular weight marker (See FIG. 78). The percent purity for reducedmajor band must be ≥97%. Visual inspection of the non-reduced referencematerial major band must yield a single broad band migrating to aposition proximal to the 97,400 Da and 116,300 Da molecular weightmarkers (FIG. 78, lane 2). The percent purity of the reference materialmajor band must be ≥97%. Visual inspection of the non-reduced referencematerial major band must yield a single broad band migrating to aposition proximal to the 97,400 Da and 116,300 Da molecular weightmarkers (FIG. 78, lane 2). The percent purity of the reference materialmajor band must be ≥97%.

Example 57 An HPLC Method for the Quantitative Determination of TritonX-100 in CTLA4^(A29YL104E)-Ig

Triton X-100 is determined at low parts per million (ppm) level (<10ppm) in protein samples of CTLA4^(A29YL104E)-Ig by HPLC. The methodinvolves extraction of Triton X-100 onto solid phase extraction mediafollowed by washing with water to remove residual protein and elution ofthe Triton X-100 with acetonitrile. The acetonitrile eluate ischromatographed using a Phenomenex Hypersil Cl column and a mobile phaseconsisting of acetonitrile: water (80:20). Detection is by UV at 225 nm.The method is linear between 1-22 ppm with the limit of detection being0.25 ppm. CTLA4^(A29YL104E)-Ig, a potential immunosuppressant agent, isa second generation fusion protein, consisting of the ligand bindingdomain of cytotoxic T lymphocyte antigen 4 (CTLA4) and the constantregion of human IgGl heavy chain. Triton X-100 an nonionic surfactant,is used for viral inactivation in the purification ofCTLA4^(A29YL104E)-Ig. Even though Triton X-100 is removed, residuallevels or absence of the surfactant from the protein needs to beestablished for product quality and regulatory purposes. To this end, amethod capable of detecting and quantitating trace levels of TritonX-100 was developed. Triton X-100 is extracted from the protein onto aSPE media and eluted with acetonitrile for analysis by HPLC.

Standard Preparation

Blank. Any sample or reference standard previously analyzed by thismethod and found not to contain detectable levels of Triton X-100 may beused as the blank. The protein concentration in the blank and sampleshould be similar. The blank should be run along with the sample(s).

Stock Standard Solution. Accurately weigh 10.0 ∀ 1.0 mg Triton X-100into a 100 mL volumetric and dilute to volume with water and mix. NOTE:Triton X-100 dissolves slowly in water. Examine the solution forcomplete dissolution (typically after 15 minutes) before use. TritonX-100 is more viscous than water, so undissolved amounts are visible inthe presence of water.

Working Standard Solution. Spike 25 μL of the Triton X-100 stockstandard solution into 0.5 mL of the CTLA4^(A29YL104E)-Ig blank. Mixthoroughly by vortex or other appropriate means. This working standardsolution contains approximately 5 ppm (or 5 μg/mL wt. vol.) of TritonX-100. NOTE: The standard solutions should be prepared fresh daily.

Sample Preparation. The sample is used as is. The blank should be runalong with the sample(s). Extraction of Triton X-100 from Standard andSample Solutions. The extraction steps described below are performedunder a vacuum of 3-5 inches of Hg.

Activation of the Solid Phase Extraction (SPE) Media. Lift the lid ofthe vacuum manifold and place test tubes in the rack inside themanifold. These are “waste” test tubes. Replace the lid and place SPEtubes on the vacuum manifold, making sure that there is a “waste” testtube underneath each SPE tube. Add one mL of acetonitrile to each tube,and apply vacuum to the tubes until all the acetonitrile has passedthrough the media bed. Repeat step. Concentration of Triton X-100 on theSPE Media from Standard/Sample Matrix. Into separate activated SPEtubes, pipette 0.50 mL each of the working standard solution, samples,and blank. Apply vacuum to the SPE tubes until the solution hascompletely passed through the media bed. Removal of Residual Proteinfrom the SPE Bed. Add one mL of water to each SPE tube, and apply vacuumto the tubes until the water has passed through the media bed. Repeatstep. Elution of Triton X-100 from the SPE Bed. Turn off the vacuum tothe manifold to bring the unit to normal pressure. Gently lift the lidof the manifold, with the standard and sample SPE tubes still attached.Replace the “waste” test tubes with a set of pre-labeled test tubes (forstandard, blank, and samples) to collect any Triton X-100 that may beeluted from the SPE beds in the following steps. These are the eluatetest tubes. Replace the lid of the manifold, making sure that eachsample, blank, and standard SPE bed has the respective eluate test tubeunderneath. Add 0.50 mL acetonitrile to each SPE tube, and apply vacuumto the tubes until all the acetonitrile has passed through the mediabed. Turn off the vacuum to the manifold, and lift the lid to retrievethe eluate test tubes. Place the acetonitrile eluates of the standard,samples, and blank into autosampler vials for injection into thechromatographic system.

System Suitability

Equilibrate the column/system with the mobile phase for about an hourbefore beginning injections. Obtain the chromatogram of the standardsolution. The retention time for Triton X-100 should be 5 ∀ 1 minutes.The efficiency of the column for Triton X-100, evaluated as the numberof theoretical plates, N, must be >2000 plates/column when calculatedaccording to the following equation:

$N = {16\mspace{11mu} \left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   t is the retention time of Triton X-100 peak, and w is the width        at baseline of the Triton X-100 peak obtained by extrapolating        the sides of the peak to the baseline. Make at least three        injections of the standard solution. The percent RSD of the area        counts of the last three injections should not be more than        3.0%.

Subtract the blank chromatogram from the standard and samplechromatograms and proceed with the following calculations:

${{Concentration}\mspace{14mu} {of}\mspace{14mu} {Triton}\mspace{14mu} X\text{-}100\mspace{14mu} ({ppm})} = {\frac{{area}\mspace{14mu} {of}\mspace{14mu} {sample}}{{area}\mspace{14mu} {of}\mspace{14mu} {standard}} \times {{wt}.\mspace{14mu} {of}}\mspace{14mu} {Triton}\mspace{14mu} X\; 100\mspace{14mu} ({mg}) \times 0.5\mspace{14mu} {ppm}}$  Where:$\mspace{20mu} {0.5 = {\frac{1000\mspace{14mu} {µg}}{1\mspace{14mu} {mg}} \times \frac{1}{100\mspace{14mu} {mL}} \times \frac{25\mspace{14mu} {microliters}}{0.5\mspace{14mu} {mL}} \times \frac{1\mspace{14mu} {mL}}{1000\mspace{14mu} {microliters}}}}$

Example 58 Assay for Determining CTLA4-Ig Composition CHO Cellular DNAContent

This procedure was developed to detect residual CHO DNA in samples ofCTLA4-Ig drug substance using a real-time quantitative polymerase chainreaction (qPCR) assay. PCR is the replication of a mixture of DNA. Thisassay used a fluorogenic probe to detect a specific CHO PCR product asit accumulates during the assay. The rate of amplification of the PCRproduct is directly proportional to the amount of starting DNA presentin the sample. The objective is to detect the amount of CHO genomic DNAin samples of CTLA4-Ig compositions.

Sample is analyzed by detecing CHO DNA in Biological Samples byQuantitive Polymerase Chain Reaction Analysis. Calculations are carriedout and results are reported to two significant figures in the units ofpg/mg. If the results are less than the quantitation limit of the assay,the results are reported as <Q.L. and the Q.L. of the assay is recorded.There is a conversion of DNA Value to picogram/milligram. The pg/mLvalue is divided by the concentration of the sample, determined by theabsorbance at 280 nm. Example calculation for a sample having a proteinconcentration of 50 mg/mL and a reported value from Bioreliance=0.67fg/μL. Step 1: Conversion to pg/mL=0.67 pg/mL Step 2: Conversion topg/mg=(0.67 pg/mL/50.0 mg/mL)=0.013 pg/mg.

Example 59 Determination of MCP-1 Protein in Biological Samples and inCTLA4-Ig Compositions

The example sets out methods used to quantitate residual MCP-1-likeprotein in biological samples and samples of CTLA4-Ig.

Materials:

Goat Anti-mouse MCP-1 (anti- R&D Systems, (Catalog No. AB- murine JE[MCP-1]) Neutralizing 479-NA), Store at −20° C. Antibody Rabbit Anti-ratMCP-1 Pepro Tech, (Catalog No. 500-P76), Store at −20° C. GoatAnti-rabbit IgG (H + L) HRP Southern Biotech (Catalog No. Conjugated4050-05, Store at 2-8° C.

Reagents

Phosphate Buffered Saline (PBS, 10 mM Phosphate Buffer, 137 mM NaCl, 2.7mM KC1, pH 7.3 to 7.5). Prepare according to manufacturer's directionson the bottle. To a vessel of sufficient size, add HPLC Grade water, PBSpellets and a stir bar. On a stirring plate mix until the pellets andsalt are dissolved. Adjust pH 7.3 to 7.5 as necessary with SodiumHydroxide or Hydrochloric Acid. Stir until well mixed. Filter through adisposable 0.22 μm filter unit. Store the solution at 2-8° C. for up to30 days from the date of preparation.

MCP-1 (Monocyte chemotactic protein-1) is a human protein which plays arole in the recruitment of monocytes to sites of injury and infection. Aprotein can be considered MCP-1-like based on homology andcross-reactivity with antibodies against MCP-1. Since the antibodiesused in the ELISA only recognize a portion of the target protein,truncated forms of MCP-1 may react in the assay even though they may notbe technically active. Thus any variant of hamster MCP-1 which reacts inthe assay will be quantified so that the assay detects full-length MCP-1and any variants of MCP-1 which contain the correct epitopes. Note thatby using a polyclonal antibody mixture, it is more likely that a numberof epitopes will be represented.

Wash Solution (1.0 mM Phosphate Buffer, 137 mM NaCl, 0.27 mM KC1, pH 7.3to 7.5 containing 0.01% v/v Tween 20). To 4.0 L of Distilled or HPLCGrade water, add a stir bar, 2 PBS tablets, 28.8 g of NaCl, and 0.4 mLof Tween 20. Mix gently until all contents are dissolved. Adjust pH 7.3to 7.5 as necessary with Sodium Hydroxide or Hydrochloric Acid. Store at2-8° C. for up to 30 days from the date of preparation.

Diluent (PBS, pH 7.3 to 7.5 containing 1% w/v BSA, and 0.05% v/v Tween20). (Please note this is an alternative to the use of commerciallyavailable diluent). To 4 L Phosphate buffered saline, add a stir bar, 40g of BSA and 2 mL of Tween 20. Mix gently until all contents aredissolved. Filter through 0.22 μm filter. Store at 2-8° C. for up to 30days from the date of preparation when stored unopened or 7 days afteropening.

Plate Coating Reagent. Reconstitute several vials of goat anti-mouseMCP-1 antibody as per manufacturer's instructions, mix by gentlyinverting several times, and combine. Prepare aliquots and store at −20°C. for up to one year (not to exceed the manufacturer's expirationdate). Avoid repeated freeze thaw. Alternatively, the lyophilized samplevials can be stored at −20° C. for greater than six months. Uponreconstitution, the antibody can be stored at 2-4° C. for one month.

Secondary Reagent. Reconstitute one vial of rabbit anti-rat MCP-1antibody as per manufacturer's instructions, mix by gently invertingseveral times. The solution is stored at 2-8° C. for up to four weeks.Alternatively the lyophilized sample vials are stable at −20° C. forgreater than one year.

Tertiary Reagent. Pool several vials of goat anti-rabbit IgG (H+L) HRPand mix by gently inverting several times. Prepare aliquots and store at−20° C. for up to 2 years (not to exceed the manufacturer's expirationdate). Avoid repeated freeze thaw. Once thawed, the vial can be storedat 2 to 8° C. for up to 30 days. [001660] Preparation of Standards. Fromthe MCP-1-like Protein stock, prepare a 25.6 ng/mL solution in diluentand dilute from there in serial dilution (2 fold) steps to 12.8, 6.4,3.2, 1.6, 0.8, and 0.4. The 0 ng/mL standard is undiluted diluent.

-   -   Example for preparing Standards using MCP-1 Reference material        with a stock concentration of 0.97 mg/mL:        -   b) (Solution A) Prepare 2.5 mL of 5200 ng/mL in diluent:            13.4 uL stock+2.487 mL diluent        -   c) (Solution B) Prepare 3.9 mL of 25.6 ng/mL in diluent:            19.2 uL stock a+3.881 mL diluent    -   Prepare remaining standards using the volumes defined below:

Final Working Concentration Solution Volume (mL) Diluent (mL) (ng/mL)25.6 ng/mL 1 mL 0 25.6 ng/mL 25.6 ng/mL 1 mL 1 mL 12.8 ng/mL 12.8 ng/mL1 mL 1 mL 6.4 ng/mL 6.4 ng/mL 1 mL 1 mL 3.2 ng/mL 3.2 ng/mL 1 mL 1 mL1.6 ng/mL 1.6 ng/mL 1 mL 1 mL 0.8 ng/mL 0.8 ng/mL 1 mL 1 mL 0.4 ng/mL

-   -   Mix each solution by vortexing at medium setting for        approximately 2-3 seconds and then gently inverting 2-3 times.

Preparation of Quality Control Samples. Prepare Quality Control (QC)samples from a vial of MCP-1 like protein in diluent. Prepare a 520ng/mL solution stock concentration from which the following qualitycontrol samples for freezing are made: 11.0 ng/mL, 5.3 ng/mL, and 1.2ng/mL. Dilute as described in the table below.

-   -   Example for preparing QCs using MCP-1 Reference material with a        stock concentration of 0.97 mg/mL:        -   a) (Solution A) Prepare 2.5 mL of 5200 ng/mL in diluent:            13.4 uL stock+2.487 mL diluent        -   b) (Solution B) Prepare 5.0 mL of 520 ng/mL in diluent: 500            uL stock a+4.500 mL diluent        -   c) Prepare final dilution of MCP-1 like protein QC samples            as follows:

Concentration QC Solution B Amount of Total QC of MCP-1 to Add Diluentto Add Volume Identification (ng/mL) (μL) (mL) (mL) QC 1 11.0 106 4.8945.0 QC 2 5.3 53 5.147 5.2 QC 3 1.2 12 5.188 5.2

Mix each solution by vortexing at medium setting for approximately 2-3seconds and then gently inverting 2-3 times.

Preparation of Test Samples. Each test sample consists of diluted testarticle. Test samples are prepared by adding 200 μL of the test sampleto 200 μL of diluent. Mix each solution by vortexing at medium speed forapproximately 2-3 seconds and then gently inverting 2-3 times.

Procedure. Plate Coating. Dilute goat anti-mouse MCP-1 antibody to 5μg/;mL with PBS. Mix the antibody and PBS solution by vortexing atmedium setting for approximately 2-3 seconds and then gently inverting2-3 times. Using a multichannel pipette, add 100 μL of this solution toeach well. Cover plates with plate sealers and incubate at roomtemperature for 12 to 18 hours. Plate Washing. Wash plates three timeswith Wash Solution using a plate washer. Set washer at three washes, 300μL/well with a zero soak time. Alternatively, plates may be washedmanually. Add 300 μL of Wash Solution to each well of each plate using amultichannel pipettor. Empty wells by flicking out into a sink and blotgently on paper towels. Repeat three times. Plate Blocking. Add 300 μLCSBB to each well using a multichannel pipettor. Incubate the plates forapproximately 60±10 minutes at room temperature. Addition of Samples.Add reference standards, quality controls (two sets of eachconcentration), and test samples to the plate, in triplicate, 100μL/well, and incubate for approximately 60±10 minutes at roomtemperature. Do not use outer wells. Prepare secondary rabbit anti-ratMCP-1 antibody. Dilute rabbit anti-rat MCP-1 antibody stock to 2 μg/mLor appropriate dilution (as per lot equivalency testing or COA) indiluent in sufficient volume for plates used in the assay. Vortex thesolution approximately 2-4 seconds at medium speed. Repeat platewashing, but wash five times. Using a multichannel pipette, add 100 μLof the secondary antibody solution to each well and incubate for 60±10minutes at room temperature. Prepare tertiary goat anti-rabbit HRPconjugate solution (0.5 μg/mL or appropriate dilution) using diluent.Mix HRP conjugate by vortexing at medium setting for approximately 2-3seconds and then gently inverting 2-3 times. Dilute to the concentrationestablished as per departmental SOP for reagent qualification. Repeatplate washing five times. Using a multichannel pipette, add 100 μL HRPconjugate to each well and incubate at room temperature in the dark forapproximately 30±5 minutes. Repeat plate washing five times. Using amultichannel pipette, add 100 μL of TMB to each well and incubate atambient temperature in the dark for approximately 2.5-4 minutes. Using amultichannel pipette, stop the TMB reaction by adding 100 μL of 1.0NH₂SO₄ in the same order as the addition of TMB was made. Read theoptical densities at 450 nm with a reference wavelength of 630 nm on a96-well plate reader.

Exemplary values. The exemplary values for the standards applies tothose values at or above the Quantitative Limit (QL) (0.8 ng/mL), asvalues below the QL are used only to help establish the extremes of thecurve. The mean, using the calculation below, background (the zero ng/mLstandard) must be ≤0.1 absorption units.

$\frac{X_{1} + X_{2} + {X_{3}\mspace{14mu} \ldots \mspace{14mu} X_{n}}}{N}$

-   -   Where: X_(1,2,3)=a specific value in a set of data        -   N=number of values in a set of data

Each standard concentration used to determine the standard curve,excluding the zero, 0.4 and the QL (0.8 ng/mL), must be within 15% ofthe target (nominal) value. The coefficient of variance (% CV) of thetriplicate AB₄₅₀ values at each standard concentration used to determinethe standard curve, with the exception of the zero, 0.4 and the QL (0.8ng/mL), must be ≤15%. The mean calculated MCP-1 concentration for a testsample of CTLA4-Ig composition should be ≤11.0 ng/mL. If the meancalculated MCP-1 concentration is ≥0.8 ng/mL (the assay QL) calculatethe % CV of the triplicate determinations and the exemplary values mustbe ≤20%. If the exemplary values are met, the mean MCP-1 concentrationis used to compute ppm. If the calculated MCP-1 concentration is ≤0.8ng/mL (assay QL) the sample is reported as <QL and the value “<0.8ng/mL” is used in computation of ppm. The % CV of the triplicatedetermination is not considered.

A QC sample is defined as a set of three wells at the statedconcentration, therefore, for the three nominal concentrations stated inthis method there are a total of six QC samples. The concentrations ofat least two of the three wells for a QC sample must be with 20% of thestated nominal concentration for the QC sample to be acceptable. Atleast twelve of the eighteen QC sample determinations must be within 20%of their respective target values. Six of the eighteen QC samples (notthree at the same concentration) may deviate more than 20% from therespective nominal value. At least four of the six QC samples must beacceptable. Two are permitted to be unacceptable, provided that they arenot both at the same concentration.

Data Evaluation. Use the Softmax™ Software with the protocol fileMCP-1.ppr to construct a standard curve using an unweighted fourparameter regression of the form.

${Absorbency}_{450} = {\frac{A - D}{1 + \left( \frac{x}{c} \right)^{b}} + D}$

-   -   Where:    -   A=Absorbency₄₅₀ value corresponding to the minimal asymptote    -   D=Absorbency₄₅₀ value corresponding to the maximal asymptote    -   c=Concentration corresponding to one half the absolute        difference between the maximal and minimal asymptotic values    -   b=the approximated slope of the linear portion of the curve    -   x=concentration of reference standard

Calculations for the Concentration of MCP-1-Like Protein. The amount ofMCP-1 like protein in the test sample may be calculated using theSoftmax Plate Reader Software. The example below illustrates how finalresults may be reported. A two fold dilution of each sample is assayed.The calculated concentration is multiplied by the appropriate dilutionfactor (2) to give the concentration in the undiluted test article.

-   -   Examples:    -   1) Diluted test sample is 10.0 ng/mL.        -   MCP-1 like protein concentration in undiluted test article            is:        -   10 ng/mL×2=20 ng/mL MCP-1    -   2) Diluted sample result is “<0.8 ng/mL”.    -   MCP-1 like protein concentration in undiluted test article is:        -   “<0.8 ng/mL”×2=“<1.6 ng/mL” MCP-1

To report the final result in ng MCP-1-like protein per mg of sample(ppm), divide the result obtained above by the undiluted sampleconcentration.

-   -   Examples:    -   1) Test sample protein concentration=50 mg/mL        -   MCP-1 like protein concentration=200 ng/mL:        -   200 ng/mL MCP-1/50 mg/mL=4 ng/mg        -   Sample is reported as 4 ppm (parts per million)    -   2) Test sample protein concentration=50 mg/mL        -   MCP-1 like protein concentration=“<1.6 ng/mL”:        -   “<1.6 ng/mL” MCP-1/50 mg/mL=“<0.032 ng/mg”        -   Sample is reported as “<QL,(<0.032 ppm)”

Example 60 Assay for determination residual levels of CD-CHO1 protein byELISA for release testing of CTLA4-I2 drug substance material

Carbonate Buffer. To a suitable vessel containing a stir bar add; 200 mLwith HPLC grade water, contents of 2 carbonate buffer capsules. Using astir plate, mix until material is in solution. Using a pH meter adjustpH to 9.6 as necessary with either 1N NaOH or H₂SO₄ Using a stir plate,mix solution for a minimum of 5 minutes. Filter solution through a 0.22μm filter. Store solution at 2-8° C. for up to 30 days and label as perdepartment procedures.

Wash Buffer (PBS containing 0.05% v/v Tween 20, pH 7.4). To a 4 L Bottleof HPLC grade water add, a stir bar, 20 PBS tablets, Add 2 mL of Tween20. Using a stir plate, mix until material is in solution. Using a pHmeter adjust pH to 7.4 as necessary with either 1N NaOH or 1N HCl. Usinga stir plate, mix solution for a minimum of 5 minutes. Store solution at2-8° C. for up to 30 days and label as per department procedures.

Streptavidin-HRP. Add 0.5 mL of HPLC grade water to a vial ofStreptavidin-HRP. To mix, cap vial and vortex gently for approximately10 seconds. Add 0.5 mL of glycerol to the vial of Streptavidin-HRP. Mix. Check solution clarity by drawing solution into a clean pasteurpipette. If solution is not clear, mix as per 3.3.2 and repeat 3.3.5until solution is clear. Determine the appropriate dilution scheme to beused for the lot of Streptavidin HRP according to department procedures.Aliquot 20 μL volumes to 0.5 mL screw cap tubes. Cap and place in a CellStorage Box. Store solution at-20° C. for up to 365 days and label asper department procedures.

Stop Solution (1 N H₂SO₄). In a fume hood, place a suitable containeronto a stir plate. Add a stir bar. Add 485.6 mL HPLC grade water. Turnon stir place to start stirring of water. Slowly add 14.4 mL ofconcentrated H₂SO₄. The solution is stable for 90 days when stored atroom temperature. Label the solution as per department procedures.

Procedure. Plate Coating. Prepare an 8 μg/mL solution of purified rabbitanti-CD CD CHO1 antibody in Carbonate Buffer to be used for coatingmicrotiter plates (10 mL of solution is required per microtiter plate).Add 100 μL of this solution to each well of an Immulon 4 microtiterplate using a multichannel pipettor. Cover the microtiter plate withparafilm and incubate at 2-8° C. for 18±2 hours.

Plate Washing. Wash plate three times with 300 μL Wash Buffer usingplate washer instrument. (Alternatively, plate may be washed manuallyusing a multichannel pipettor.) Following the last wash, the plateshould be turned upside down and tapped against a paper towel laid on ahard surface.

Plate Blocking. Using a multichannel pipete, add 300 μL SeaBlock to eachwell. Incubate the plate in the dark for 1 hour (±6 minutes) at roomtemperature. Plates may either be wrapped with aluminum foil or placedin a cabinet or drawer. Preparation of Standard Curve samples usingTeknova Diluent in 15 mL graduated sterile polypropylene tubes accordingto the dilution scheme below. Note: The dilution scheme below is for 5plates. Adjust volumes as necessary for the number of plates in theassay. Obtain protein concentration of the CD-CHO1 Protein ReferenceStandard (Ref Std) from the Certificate of Analysis (COA). Prepare a 30μg/mL solution of CD-CHO1 Protein Ref Std. Calculate required volume ofCD-CHO1 Protein Ref Std to obtain a 30 μg/mL solution. Minimum transfervolume for the CD-CHO1 Protein Ref Std should be 10 μL.

Formula:${Volume} = \frac{{Desired}\mspace{14mu} {Concentration} \times {Desired}\mspace{14mu} {Volume}}{{Reference}\mspace{14mu} {Material}\mspace{14mu} {Concentration}}$Note:  Ensure  units  are  compatible.Example:$\frac{30\mspace{14mu} {µg}\text{/}{mL} \times 10\mspace{14mu} {mL}}{25\mspace{14mu} {mg}\text{/}{mL}} = {12\mspace{14mu} {µL}}$

Calculate volume of diluent by subtracting the required volume ofCD-CHO1 Protein Ref Std from the desired volume.

(Desired Volume)−(Ref Std volume)=(Diluent volume)   Formula

10.0 mL-0.012 mL=9.988 mL Diluent   Example

Add calculated volume of CD-CHO1 Protein Ref Std to a 15 mL sterile tubewhich contains calculated diluent volume. Cap tube and vortex at asetting between for 2-4 seconds. Prepare remaining standards. The tablebelow is shown as an example:

Working Total Concentration Volume Diluent Volume Concentration (ng/mL)(mL) (mL) (mL) (ng/mL) 30,000 0.6 5.4 6.0 3,000 3,000 2.0 4.0 6.0 1,0001,000 2.0 4.0 6.0 333.3 333.3 2.0 4.0 6.0 111.1 111.1 2.0 4.0 6.0 37.037.0 2.0 4.0 6.0 12.3 12.3 4.0 2.0 6.0 8.2 NA NA 4.0 4.0 0

Cap tube following each dilution step and vortex gently for 2-4 secondsprior to proceeding to next dilution step.

Prepare Quality Control (QC) solutions using Teknova Diluent in 15 mLgraduated sterile polypropylene tubes according to the dilution schemebelow. Obtain protein concentration of the CD-CHO1 Protein ReferenceMaterial (Ref Mat) from the Certificate of Analysis (COA). Prepare a 30μg/mL solution of CD-CHO1 Protein Reference Standard (Ref Mat). Cap tubeand vortex at a setting between for 2-4 seconds. Prepare QC solutions atthe concentrations of 700, 100, and 25 ng/mL. An example dilution schemeis shown below:

Working Total Concentration Volume Diluent Volume Concentration (ng/mL)(mL) (mL) (mL) (ng/mL) QC 1 30,000 0.233 9.767 10.0 700 QC 2 700 1.4308.570 10.0 100 QC 3 100 2.500 7.500 10.0 25

Cap tube for each QC solution and vortex gently for 2-4 seconds. QualityControl solutions can be prepared fresh on the day of the assay or theycan be aliquotted and frozen. Determine the actual concentration of thethree QC solutions in three independent experiments. Average the resultsfrom the three experiments for each QC solution and issue a COA for eachof the three QC solutions. These experimentally determined QCconcentrations are to be used as target values when performing analysison CTLA4-Ig samples. Store QC solutions in ready to use aliquots at −70°C. QC solutions expire 6 months after preparation. Remove QC solutionsfrom storage on the day of assay and thaw at room temperature. Vortexthawed QC solutions at medium speed for 2-4 seconds before use.

Sample Preparation. Prepare approximately a 12.5 mg/mL solution for eachCTLA4-Ig sample to be analyzed in diluent by adding 250 μL drugsubstance sample to 750 μL diluent. Cap tube and vortex gently for 2-4seconds. Prepare a 6.25 mg/mL solution for each CTLA4-Ig sample to beanalyzed by adding 400 of the 12.5 mg/mL solution to a tube containing400 μL of diluent. Cap tube and vortex gently for 2-4 seconds. Prepare a3.125 mg/mL solution for each CTLA4-Ig sample to be analyzed by adding400 of the 6.25 mg/mL solution to a tube containing 400 μL of diluent.Cap tube and vortex gently for 2-4 seconds. Wash Plate by repeating washstep. Add 100 μL per well of each standard concentration, samples, andQC solutions in triplicate to the blocked and washed plate. Each QCsolution is added twice to a total of six wells per plate. Incubate for1 hour in the dark (±6 minutes) at room temperature. Repeat wash 5times. Remove Streptavidin-HRP from freezer and allow to come to roomtemperature. Dilute rabbit anti-CD CHO1-Biotin antibody to 2 μg/mL inTeknova Buffer. Vortex the solution approximately 2-4 seconds at mediumspeed. Using a multichannel pipette, add 100 μL per well. Incubate for 1hour (±6 minutes) in the dark at room temperature. DiluteStreptavidin-HRP to the concentration established for use in the assaybased on the qualification of the reagent lot as per departmentprocedure. Cap and vortex the solution approximately 2-4 seconds atmedium speed. Using a multichannel pipette, add 100 μL ofStreptavidin-HRP dilution to each well. Incubate at room temperature inthe dark for 1 hour (±6 minutes). Repeat. Using a multichannel pipetteadd 100 μL of TMB chromogen to each well. Incubate at ambienttemperature for 2 minutes (±12 seconds). Using a multichannel pipette,add 100 μL/well of Stop Solution (1 N H₂SO₄). Add Stop Solution in thesame order to plates and wells as the chromogen was added to ensure thesame reaction times of chromogen with the enzyme in each well. Using aSpectraMax Plus plate reader, measure absorbance at 450 nm with areference wavelength of 630 nm on an appropriate 96 well plate reader.

Data Evaluation. Refer to the Softmax program template as it generatesmean, standard deviations and % CVs, etc. Determine each exemplaryvalues using the triplicate absorbance values obtained for eachreference, QC and sample dilutions assayed. Generate Standard Curve.Model reference standard data using a four-parameter regression of theform.

${AB} = \frac{\min - \max}{1 + \left( \frac{C}{{ED}_{50}} \right)^{B}}$

-   -   Where:    -   AB=Absorbency at 450 nanometers    -   A=Absorbency value corresponding to the minimal asymptote    -   D=Absorbency value corresponding to the maximal asymptote    -   c=Concentration corresponding to one half the absolute        difference between the maximal and minimal asymptotic values    -   B=the approximated slope of the linear portion of the curve    -   x=Concentration of CD-CHO1 reference material

Determine the coefficient of determination (R²) of the regression linefor the standards using the calculated mean using the formula above.Calculation of CD-CHO1 concentration in samples. Multiply the meansample results by the appropriate dilution factor (i.e. 4, 8, and 16) toobtain the concentration of CD-CHO1 in the original sample in ng/ml.Divide the results by the reported CTLA4-Ig protein concentration(mg/ml) to obtain the concentration of CD-CHO1 in ng/mg of CTLA4-Ig.Determine the mean of the all of the results, which fall within therange of the standard curve. Calculate the CD-CHO1 protein concentrationin the undiluted sample by applying the dilution factor. To determinethe CD-CHO1 protein concentration relative to the CTLA4-Ig sampleconcentration divide by the undiluted concentration.

Examnle Calculation:

$\frac{{Mean}\mspace{14mu} {Sample}\mspace{14mu} {Result}}{{BMS} - {188667\mspace{14mu} {Concentration}}} = {\frac{235\mspace{14mu} {ng}\mspace{14mu} {CD}\mspace{14mu} {CHOP}\text{/}{mL}}{50\mspace{14mu} {mg}\text{/}{mL}} = {4.7\mspace{14mu} {ng}\text{/}{mg}}}$

Note: ng CD CHO1 mg product (ng/mg) is equivalent to parts per million(ppm).

Exemplary values. Exemplary values for the Standards. The coefficient ofdetermination (R²) for the Standard Curve should be ≥0.99. The meanbackground for the zero ng/mL Standard should be 0.10 absorption units.The mean of the calculated values (ng/mL) at each standard concentrationused to determine the Standard Curve, excluding zero and concentrationsbelow QL (12.3 ng/mL), must be within 20% of the target (nominal) value,as determined by the software. The coefficient of variation (% CV) ofthe triplicate absorbance values at each Standard concentration used todetermine the Standard Curve, excluding zero and concentrations below QL(12,3 ng/mL), must be less than 20%, as determined by the software. Toensure that at lease two congruent data points are available forcalculation, the standards, quality controls, and samples are loaded intriplicate wells. Analyze each triplicate value separately. Drop thevalue that lies furthest from the target.

-   -   Example:

Target Value (ng/mL) Actual Value (ng/mL) 50 25 48 49

The single value that is furthest from the target value of 50 ng/mL is25 ng/mL. By eliminating the 25 ng/mL value from the triplicate, themean of the remaining values meet all of the exemplary values.

If it is shown that the mean of the remaining two values still do notmeet the exemplary values, then the single point is eliminated and thecurve is recalculated.

-   -   Example:

Target Value (ng/mL) Actual Value (ng/mL) 50 10 10.5 25

The mean value is >20% from the target regardless of which value iseliminated, therefore, the single point is dropped from the curve andthe curve will be recalculated. Only 2 points on the standard curve maybe eliminated.

Exemplary values for QC Samples. A QC sample is defined as a set ofthree wells at the stated concentration, therefore, for the threenominal concentrations stated in this method there are a total of six QCsamples. At least two of the three wells for a QC sample must be within20% of the nominal for the QC sample to be acceptable. At least four ofthe six QC samples must be within 20% of their respective targetconcentrations; two of the six QC samples (not two at the sameconcentration) may exceed the 20% deviation from nominal, as calculatedby the software. Not more than 6 of the 18 QC sample wells may deviatemore than 20% of the respective target concentrations.

Exemplary values for Test Samples. The average absorbance for the testsample assayed must be less than the highest point on the standardcurve. If the average absorbance of the sample exceeds the averageabsorbance 3000 ng/mL standard, the test sample must be dilutedsufficiently so as to obtain a mean absorbance between 3000 and 12.3ng/mL. The average absorbance of at least one of the three sampledilutions (12.5, 6.25 and 3.125 mg/ml) must fall within the range of thestandard curve for a reportable result, unless the average absorbancefor all dilutions are below the average absorbance of the QL (12.3 ng/mLstandard). In that case the test samples are reported as <QL. The meanof the triplicate absorbance values of the Sample dilutions that aregreater than the QL and fall within the range of the standard curve mustexhibit a CV of less than 20%, as determined by the software. If uponelimination of one of the triplicate absorbance and recalculation the CVis still >20% or if two Sample dilutions have CV's for the absorbancegreater then 20% and fall within the range of the standard curve theanalysis this Sample must be repeated.

Example 61 Assay for Residual Amount of Triton X-100 in CTLA4-IgComposition

Materials

HPLC Vials Waters, Total Recovery Vial Kit, Screw Cap with bondedpreslit PTFE/Silicone Septa (Catalog 186000385) Note: Glass vials arerequired Solid Phase Extraction Waters OASIS HLB, 30 mg/lcc, Tubes(Catalog No. WAT094225) Column Thermo Electron Corp. SAS Hypersil, 5μ,4.6 × 250 mm (Part Number 30505-254630)

Instrumentation

Liquid Chromatograph Waters 2695 Separations Module Detector Waters 2487Dual Wavelength UV Detector SPE Column Processor JT Baker VacuumManifold, Model Spe-21 Analytical Balance Any balance capable ofaccurately weighing 0.01 mg Integration Waters Empower Data System

Preparation of Reagents

Mobile Phase Preparation: Acetonitrile: Water(80:20). For 1 L,thoroughly mix with a stir bar, 800 mL of acetonitrile and 200 mL ofpurified or HPLC grade water. Filter through a 0.2 μm nylon filter.Degas mobile phase using an inline degasser such as an Alliance system,or using helium sparge. Prepare fresh daily.

2N NaOH. Weigh, and quantitatively transfer 80±1 g of solid NaOH to a 1L flask. Bring to volume with purified or HPLC grade water. Mix wellwith stir bar and filter through a 0.22 μm filter apparatus.Alternatively, serial dilute 10N NaOH solution. Store up to 6 months atroom temperature.

Drug Substance Buffer. Weigh and quantitatively transfer 3.45g NaH₂PO₄.H2O and 2.92g NaCl to a 1 L flask. Add approximately 800 mL of purifiedor HPLC grade water. Mix well with a stir bar, and bring to volume withpurified or HPLC grade water. Adjust pH to 7.5 with 2N NaOH. Filtersolution through a 0.22 μm filter unit. Store up to 4 months at 4° C.

Preparation of Standard Sample Blank. Any CTLA4-Ig sample or referencematerial previously analyzed and found not to contain detectable levelsof Triton X-100 may be used as the Sample Blank. The Sample Blankprotein should be run along with the samples(s). Triton X-100 dissolvesslowly in water. Examine the solution for complete dissolution(typically after 15 minutes) before use. Triton X-100 is more viscousthan water, so undissolved amounts are visible in the presence of water.

Preparation of 10.0 μg/mL Triton X-100 Stock Standard #1. Accuratelyweigh 10.0±1.0 mg Triton X-100 into a 100 mL volumetric flask and diluteto volume with water; and mix gently with a stir bar. Label as TX100Stock Standard A. Take 10 mL of TX100 Stock Standard A into a 100 mLvolumetric flask. Dilute to volume with water. Label as TX100 StockStandard #1. Prepare fresh daily.

Preparation of 10.0 μg/mL Triton X-100 Stock Standard #2. Accuratelyweigh 10.0±1.0 mg Triton X-100 into a 100 mL volumetric flask and diluteto volume with water; and mix gently with a stir bar. Label as TX100Stock Standard B. Take 10 mL of TX100 Stock Standard B into a 100 mLvolumetric flask. Dilute to volume with water. Label as TX100 StockStandard #2. Prepare fresh daily.

Preparation of TX100 System Suitability Evaluation Solution #1 (SS#1).From TX100 Stock Standard #1 prepare a 5.0 μg/mL TX100 SystemSuitability Evaluation Solution by diluting 300 μL of TX100 StockStandard #1 with 300 μL of acetonitrile. Mix well by pipetting up anddown.

Preparation of TX100 System Suitability Evaluation Solution #2 (SS#2).From TX100 Stock Standard #2 prepare a 5.0 μg/mL TX100 SystemSuitability Evaluation Solution by diluting 300 μL of TX100 StockStandard #1 with 300 μL of acetonitrile. Mix well by pipetting up anddown.

Preparation of TX100 Pass Control, Limit Standard, Fail Control. Fromthe TX Stock Standard #1, prepare the solution identified in Tablebelow.

Solution Identification Dilution Procedures Pass Control (0.4 μg/mL)Dilute 20 μL with 480 μL of CTLA4-Ig DS Limit Standard (0.5 μg/mL)Dilute 25 μL with 475 μL of CTLA4-Ig DS Fail Control (0.6 μg/mL) Dilute30 μL with 470 μL of CTLA4-Ig DS

Preparation of Sample. The sample is used without concentration ordilution. Procedure: Extraction of Triton X-100 from Standard and SampleSolution. CAUTION: The extraction steps described below are performedunder a vacuum of 3-3.5 inches Hg.

Activation of the Solid Phase Extraction (SPE) Media. Lift the lid ofthe vacuum manifold and place empty 12×75 mm test tubes in the rackinside the manifold. These are “waste” test tubes. Replace the lid andplace SPE cartridges on the vacuum manifold, making sure that there is a“waste” test tube underneath each SPE tube. Add 1000 μL of acetonitrileto each SPE cartridge, and apply vacuum until all the acetonitrile haspassed through the media bed. Repeat with an additional 1000 μL ofacetonitrile. Add 500 μL of purified or HPLC grade water to each SPEcartridge and apply vacuum until all water has passed through the mediabed. Repeat with an additional 500 μL of purified or HPLC grade water.

Concentration of Triton X-100 on the SPE Media for the Limit Standardand Controls. Pipette 500 μL each of the Limit Standard, Pass Control,Fail Control Blank, and samples into separate, activated SPE cartridges.Apply vacuum to the SPE tubes until each solution has completely passedthrough the media bed.

Removal of Residual Protein from the SPE Bed. Add 1000 μL of water toeach SPE cartridge, and apply vacuum to the tubes until the water haspassed through the media bed. Repeat step with an additional 10004 ofwater.

Elution of Triton X-100 from the SPE bed Turn off the vacuum to themanifold to release the unit pressure to zero. Gently lift the lid ofthe manifold, with the SPE cartridges still attached. Replace the“waste” test tubes with a set of pre-labeled “eluate” test tubes orautosampler vials (prelabeled limit standard, pass control, failcontrol, blank, and samples) to collect any Triton X-100 that elutesfrom the SPE beds. Replace the lid of the manifold, making sure thateach limit standard, pass control, fail control, blank, and samples SPEcartridge has a respective eluate test tube, or autosampler vialunderneath. Add 500 μL acetonitrile to each SPE cartridge, and applyvacuum to the tubes until all the acetonitrile has passed through themedia bed. Turn off the vacuum to the manifold, and lift the lid toretrieve the eluate test tubes, or autosampler vials. If using eluatetest tubes, place the acetonitrile eluates of the limit standard, passcontrol, fail control, blank, and samples into autosampler vials forinjection into the chromatographic system. If using autosampler vials tocollect the eluate, place the vials directly into the chromatographicsystem.

Run Conditions

Wavelength 225 nm Sensitivity 0.1 AUFS Mobile Phase Acetonitrile:water(80:20) Flow Rate 0.8 mL/min Injection Volume 25 μL Column TemperatureAmbient (20-25° C.) Approximate Retention Time Triton X-100: 5.0 ± 1minute Total Run Time 10 minutes Autosampler Temperature 10 ± 4° C.

System Suitability. Set up the chromatographic system; allow lamp towarm up and system to equilibrate with mobile phase for at least onehour prior to analysis. Inject SS #1 a minimum of four times. Use thesecond SS #1 injection for all system suitability analyses, unlessotherwise specified. The retention time for Triton X-100 should be 5.0±1minutes. The efficiency of the column for Triton X-100, evaluated as thenumber of theoretical plates (N), must be ≥2000 plates/ column whencalculated according to the following equation:

$N = {16\mspace{11mu} \left( \frac{t}{w} \right)^{2}}$

-   -   Where:    -   t=retention time of Triton X-100 peak measured from time of        injection to time of elution of peak maximum.    -   w=width of the Triton X-100 peak measured extrapolating the        sides of the peak to the baseline.

The resolution between the Triton X-100 peak and the nearest adjacentpeak (if present) must be ≥1.

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{W_{1} + W_{2}}$

-   -   Where:    -   t=retention times of the Triton X-100 peak and the adjacent peak        in the standard.    -   W=corresponding widths of the bases of the peaks obtained by        extrapolating the sides of the peaks to the baseline.

Calculate the response factors of the last three SS#1 injections usingthe following equation:

${RF} = \frac{A}{W}$

-   -   Where:    -   A=Area of Triton X-100 peak    -   W=Weight of Triton X-100 (in mg) used in the preparation of the        corresponding TX100 stock standard solution

The response factors of the last three injections of SS#1 must have arelative standard deviation (RSD) of ≤10%. Calculate the % RSD using thefollowing equation:

${\% \mspace{14mu} R\; S\; D} = {\frac{{Standard}\mspace{14mu} {Deviation}}{Mean} \times 100}$${{Standard}\mspace{14mu} {Deviation}} = \sqrt{\frac{{n\; {\sum x^{2}}} - \left( {\sum x} \right)^{2}}{n\left( {n - 1} \right)}}$

-   -   Where:    -   n=number of measurements in the sample    -   x=individual measurements

Compare the response factor of the single injection of SS#2 to theaverage response factor of the last three injections of SS#1. Thepercent difference between the SS#2 response factor and the averageresponse factor of the three SS#1 injections must be 10%. Calculate thepercent difference using the following equation.

${\% \mspace{14mu} {Difference}} = {{\left\lbrack \frac{{RF}_{1} - {RF}_{2}}{{RF}_{1}} \right\rbrack \times 100}}$

-   -   Where:    -   RF1=Average response factor of the three SS#1 injections    -   RF2=Response factor of SS#2

Make a single injection of Sample Blank post-SPE. If Triton X-100 levelsare found, make two more injections of the Sample Blank. If Triton X-100is present discard the CTLA4-Ig Drug Substance and make new LimitStandard, and Controls with a new lot of CTLA4-Ig Drug Substancepreviously analyzed and found to contain no detectable levels of TritonX-100. If the above exemplary values are not met, extend theequilibration of the column for another hour and reinject the standardsolution. If the exemplary values are still not met, execute thefollowing steps. Check for leaks making sure all tubing connections aresecure. If extended equilibration does not work, adjust the organiccontent of the mobile phase and/or make a new batch of the mobile phase,and re-prepare the SS#1 and SS#2. Change the column if extendedequilibration and/or mobile phase adjustment do not result in theexemplary values being met. Before repeating equilibrate for at leastone hour.

Injection Sequence. Preliminary equilibration of HPLC system andsuccessful running of above. Make a single injection of drug substancebuffer post-SPE, refer to section above as a calibration blank. Observethat there is no Triton X-100 response. If a peak is present at theretention time of the Triton X-100 peak, continue to inject the blankuntil no response is noted. Make a single injection of CTLA4-Ig drugsubstance post-SPE, refer to section above as a sample blank. Observethat there is no Triton X-100 response. This chromatogram will besubtracted from the standard, control and sample chromatograms beforedata processing is performed. Make duplicate injections of the PassControl, Limit Standard, an Fail Control. Make a single injection of thedrug substance buffer, and no triton X-100 response must be noted. Makeduplicate injections of the samples. The sample injections are bracketedby duplicate injections of Limit Standard and one injection of the drugsubstance buffer so that, there are no more than 10 sample injectionsbetween bracketing Limit Standard and Drug Substance injections. The endof the run must be completed with duplicate injections of the LimitStandard, and one injection of the drug substance buffer blank.

Data Processing. Subtract the chromatogram of the Sample Blank injectionfrom all standard, control, and sample chromatograms before proceedingwith data processing. Assure the Triton X-100 peak in the LimitStandard, Control, and sample chromatograms are properly integrated. TheTriton X-100 peak in the sample, if present, must be within the sameretention time window as the Limit Standard. Average the peak area ofeach set of duplicate injections. Duplicate injections must have a %difference in peak area of ≤10%. If a duplicate injection of the LimitStandard, Pass Control, or Fail Control do not meet this criterion, theentire run is considered invalid and will need to be repeated. If theduplicate injections of the sample fail do not meet this criterion, thesample is considered invalid and will need to be repeated. All othersamples, control, and standards are considered valid as long as theymeet the ≤10% criterion. The averaged peak area for the Triton X-100peak in all bracketing Limit Standards, including the final injections,must be within 10% of the initial averaged peak area of the LimitStandard. If any intermediate Limit Standard fails to meet the 10%comparison requirement, all samples analyzed after the last passingLimit Standard are considered invalid and must be reanalyzed.

Evaluation of Limit Standard, Pass Control, Fail Control Samples.Compare the averaged Triton X-100 peak areas for the Limit Standard,Pass Control, and Fail Control. The peak area of the Fail Control samplemust be greater than that of the Limit Standard. The peak area of thePass Control sample must be less than that of the Limit Standard. Ifboth conditions are met, continue on to the evaluation of the samples.

Evaluation of Samples. The averaged Triton X-100 peak area of the LimitStandard must be corrected to account for the amount of material weighedin the preparation of Triton X-100 Stock Standard #1, as follows:

A_(L)=A_(LS)×(10.00 mg/Wt.)

-   -   Where:    -   A_(L)=Peak area corrected to 0.5 μg/mL limit    -   A_(Ls)=Averaged Triton X-100 peak area of bracketing Limit        Standards    -   Note: A_(L) is the corrected area at the 0.5 μg/mL limit and        will be used for comparison to samples.

Compare the average Triton X-100 peak area from the sample to A_(L). Ifpeak area ≤A_(L), sample passes specification. If peak area >A_(L),sample fails specification. Report the results passing specification as“<0.50 ppm” or failing specification as “>0.50 ppm” or as otherwiserequired by reporting convention.

Example 62 Assay to Quantitate the Amount of Residual Protein A inCTLA4-I2 Drug Substance Material

Materials

Rabbit anti-Protein A Antibody Sigma, (Catalog No. P3775) Biotinylatedanti-Protein A monoclonal Sigma, (Catalog No. B3150) antibody

Reagents

Carbonate Coating Buffer. To a suitable vessel add; Add a stir bar. Add500 mL of HPLC grade, or Millipore, water. Add contents of 5 carbonatecapsules. Stir until well mixed. Check and adjust pH to 9.6±0.1 usingNaOH (1.16) or HCl (1.23). Pour solution into a 500 mL filtersterilization system. Apply a vacuum to filter sterilize the solution.Under aseptic conditions remove filter unit and cap bottle. The solutionis stable for 30 days when stored at 2-8° C. Label the solution as“Carbonate Coating Buffer”. Wash buffer: (PBS+0.05% Tween 20): To a 4 Lbottle of HPLC grade, or Millipore, water add; Add a stir bar. Add 20PBS tablets. Add 2.0 mL of Tween 20. Check and adjust pH to 7.4±0.1using NaOH or HC1. Using a stir plate, stir until well mixed. Thesolution is stable for 30 days when stored at 2-8° C. Label the solutionas “Wash Buffer”. Stop Solution (1 N H₂SO₄) or use 1.000 Normal SulfuricAcid from VWR without diluting. In a fume hood place a suitablecontainer onto a stir plate: Add a stir bar Add 485.6 mL of HPLC grade,or Millipore, water. Turn on stir plate to start stirring of water.Slowly add 14.4 mL of concentrated H₂SO₄. The solution is stable for 30days when stored at room temperature. Label the container as “StopSolution”.

Acetate Buffer (0.5M Acetic Acid, 0.1M Sodium Chloride, 0.1% Tween 20,pH 3.5). In a fume hood place a suitable container onto a stir plate:Add a stir bar. Add 400 mL of HPLC grade, or Millipore, water. Turn onstir plate to start stirring of water. Slowly add 14.8 mL ofconcentrated Glacial Acetic Acid . Stir until well mixed. Add 2.9 gSodium chloride. Stir until well mixed. Check and adjust pH to 3.5±0.1using NaOH or HCl. Add 0.5 mL Tween 20. Stir until well mixed. Adjust toa final volume of 500 mL with HPLC grade, or Millipore water. Stir untilwell mixed. The solution is stable for 30 days when stored at 2-8° C.Label the container as “Acetate Buffer.”

Rabbit anti-Protein A Antibody. Remove vial from the refrigerator andallow to come to room temperature. Add 2.0 mL of HPLC grade, orMillipore, water. Aliquot 20 μL volumes to 0.5 mL screw cap tubes. Capand place in a Cell Storage Box. The solution is stable for 365 dayswhen stored at −20° C. Biotinylated anti-Protein A Antibody Remove vialfrom the refrigerator and allow to come to room temperature. Add 1.0 mLof HPLC grade, or Millipore water. Aliquot 604 volumes to 2.0 mL screwcap tubes. Cap and place in a Cell Storage Box. The solution is stablefor 365 days when stored at −20° C.

Streptavidin-HRP. Add 0.5 mL of HPLC grade, or Millipore, water to avial of Streptavidin-HRP. To mix, cap vial and vortex gently forapproximately 10 seconds. Add 0.5 mL of glycerol to the vial ofStreptavidin-HRP. Cap vial and gently invert vial several times. Checksolution clarity by drawing solution into a clean pasteur pipette.Aliquot 20 μL volumes to 0.5 mL screw cap tubes. Cap and place in a CellStorage Box. The solution is stable for 365 days when stored at-20° C.

Preparation of Standard. Obtain protein concentration of the Protein AReference Material (ref mat) from the Certificate of Analysis (COA).Prepare a 11,500 ng/mL solution of Protein A ref mat, by thawing out thestock Protein A solution at room temperature. Calculate required volumeof Protein A ref mat to obtain a 11,500 ng/mL solution. Minimum transfervolume should be 10 μL.

$\begin{matrix}{{{Formula}\text{:}}{{Volume} = \frac{{Desired}\mspace{14mu} {Concentration} \times {Desired}\mspace{14mu} {Volume}}{{Reference}\mspace{14mu} {Material}\mspace{14mu} {Concentration}}}{{Note}\text{:}\mspace{14mu} {Ensure}\mspace{14mu} {units}\mspace{14mu} {are}\mspace{14mu} {{compatible}.{Example}}\text{:}}{\frac{2.3\mspace{11mu} {µg}\text{/}{mL} \times 10\mspace{14mu} {mL}}{0.25\mspace{14mu} {mg}\text{/}{mL}} = {92\mspace{14mu} {µL}}}} & \;\end{matrix}$

Calculate volume of Acetate buffer (3.4) by subtracting the requiredvolume of Protein A ref mat from the desired volume.

(Desired Volume)−(Ref Std volume)=(Diluent volume)   Formula

10.0 mL-0.920 mL=9.180 mL Diluent   Example

Add calculated volume of Protein A ref mat to a 15 mL sterile tube whichcontains calculated Acetate Buffer volume. Cap tube. Gently vortex(setting 4 on Vortex Genie 2) for 2-4 seconds. Prepare remainingstandards using volumes defined below:

Working Working Conc. Acetate Total Final Concentration Volume BufferVolume Concentration (ng/mL) (mL) (mL) (mL) (ng/mL) 2,300,000 (2.3mg/mL) 0.010 1.990 2.0 11,500 11,500 0.010 4.780 4.790 24.0 24.0 2.0 2.04.0 12.0 12.0 2.0 2.0 4.0 6.00 6.00 2.0 2.0 4.0 3.00 3.00 2.0 2.0 4.01.50 1.50 2.0 2.0 4.0 0.75 0.75 2.0 2.0 4.0 0.375 0.375 2.0 2.0 4.00.188 N/A N/A 2.0 2.0 0

Cap tube following each dilution step and vortex gently for 2-4 secondsprior to proceeding to next dilution step. Incubate Reference Standardand Quality Control samples for 10 minutes at room temperature beforeaddition to microtiter plate. Each Standard concentration is analyzed intriplicate wells.

Preparation of Test Samples. Prepare concentrations of 5 mg/mL, 2.5mg/mL and 1.25 mg/mL of the CTLA4-Ig test samples in polypropylene tubesusing Acetate buffer. Thaw CTLA4-Ig sample at room temperature beforeusing to prepare dilutions. Calculate required volume of test sample toobtain a 5.0 mg/mL solution. Minimum transfer volume should be 10 μL.

Formula:${Volume} = \frac{{Desired}\mspace{14mu} {Concentration} \times {Desired}\mspace{14mu} {Volume}}{{Test}\mspace{14mu} {Sample}\mspace{14mu} {Concentration}}$Example:$\frac{5.0\mspace{11mu} {mg}\text{/}{mL} \times 1.0\mspace{14mu} {mL}}{25\mspace{14mu} {mg}\text{/}{mL}} = {200\mspace{14mu} {µL}}$

5.1.3 Calculate volume of Acetate buffer by subtracting the requiredvolume of test sample from the desired volume.

(Desired Volume)−(test sample volume)=(Diluent volume)   Formula

1.0 mL-0.200 mL=0.800 mL Diluent   Example

Add calculated volume of test sample to a 15 mL sterile tube whichcontains calculated Acetate Buffer volume. Cap tube and gently vortex(setting 4 on Vortex Genie 2) for 2-4 seconds. est samples are incubatedfor 10 minutes at room temperature before adding to microtiter plate.

Preparation of Quality Control Samples. Obtain protein concentration ofthe Protein A Reference Material from the COA. Prepare a 11,500 ng/mLsolution of Protein A ref mat. Prepare Quality Control (QC) samples asdescribed in the table below.

Working Working Conc. Acetate Total Final Concentration Volume BufferVolume Concentration (ng/mL) (mL) (mL) (mL) (ng/mL) 2,300,000 (2.3mg/mL) 0.010 1.990 2.0 11,500 11,500 0.010 4.780 4.790 24.0 24.0 0.83333.166 4.0 5.0 (QC1) 5.0 1.6 2.4 4.0 2.0 (QC1) 2.0 1.0 3.0 4.0 0.5 (QC1)

Aliquot 700 μL volumes to 2.0 mL screw cap tubes. Cap and place in aCell Storage Box. The solution is stable for 180 days when stored at−70° C. or below. The Protein A concentrations in the QC samples are tobe pre-determined by: Performing 3 independent Protein A ELISA assaysusing this method. The 18 results (3 independent assays times 6 wellsper assay) will be averaged. The Protein A concentration for each QCsample will be assigned to each preparation. On the day of anexperiment, thaw one vial of each QC samples at room temperature, ormake QC samples fresh from stock vial of Protein A. Vortex gently(setting 4 on Vortex Genie 2) for 2-4 seconds.

Procedure. Plate Coating with Capture Antibody. Obtain proteinconcentration of the Rabbit anti-Protein A Antibody from themanufacturers COA. Calculate required volume of Rabbit anti-Protein AAntibody to obtain 10 mL of a 100 μg/mL solution. Minimum transfervolume should be 10 μL.

Formula:${Volume} = \frac{{Desired}\mspace{14mu} {Concentration} \times {Desired}\mspace{14mu} {Volume}}{{Antibody}\mspace{14mu} {Concentration}}$Note:  Ensure  units  are  compatible.Example:$\frac{100\mspace{14mu} {µg}\text{/}{mL} \times 3\mspace{14mu} {mL}}{25\mspace{14mu} {mg}\text{/}{mL}} = {12\mspace{14mu} {µL}}$

Calculate volume of Diluent by subtracting the required volume of Rabbitanti-Protein A Antibody from the desired volume.

(Desired Volume)−(Antibody Volume)=(Diluent Volume)   Formula

3.0 mL-0.012 mL=2.988 mL Diluent   Example

Add calculated volume of Rabbit anti-Protein A Antibody to a 15 mLsterile tube, which contains calculated diluent volume. Cap tube andvortex gently for 2-4 seconds. Prepare a 1 μg/mL solution of rabbitanti-Protein A antibody in Coating Buffer, using the formulas. Add 100μ1 of the solution to each well of an Immulon IV microtiter plate.Incubate at 2-8° C. for 18±2 hours. Wash plates three times with WashSolution using the plate washer with 300 μL wash buffer and zero soaktime. The plates may alternatively be washed using a multi channelpipette. Using a multichannel pipette add 200 μL of SuperBlock™ to eachwell. Incubate the microtiter plate in the dark for 60 minutes at roomtemperature. Repeat wash. Add 100 μL of each Reference Standard, QualityControl and test samples to assay plate. Incubate the microtiter platein the dark for 60 minutes,±10 minutes, at room temperature. Repeatwash. Obtain protein concentration of the Biotin anti-Protein A Antibodyfrom the manufacturers COA. Prepare 1.0 mL of a 1.0 mg/mL solution ofBiotin anti-Protein A Antibody in diluent. Vortex at medium speed. Add50 μL of 1 mg/mL solution (7.9.1) to 0.950 mL of diluent. Vortex atmedium speed. Add 150 μL of 50 μg/mL solution (7.9.3) to 14.850 mL ofdiluent. Add 100 μL to each well using a multichannel pipettor. Incubateat room temperature for 60 minutes±10 minutes. Repeat wash. DiluteStreptavidin-HRP as follows: Add 10 μL of Streptavidin-HRP to 0.990 μLof Diluent to yield 0.01 mg/mL Streptavidin-HRP. Vortex at medium speed.Add 80 μL of 0.01 mg/ml Streptavidin-HRP to 39.920 mL of Diluent. Vortexat medium speed. Add 100 μL to each well using a multichannel pipettor.Incubate for 30 minutes,±5 minutes, at room temperature. Remove TMB fromrefrigerator and decant a minimum of 10 mL per plate of TMB into asuitable container. Place in a dark location and allow to come to roomtemperature. Repeat wash, but wash five times. Add 100 μL TMB chromogento each well. Incubate at room temperature for approximately 2minutes,±1 minute. Stop chromogen reaction by adding 100 μL/well of StopSolution. Add Stop Solution in the same order to plates and wells as thechromogen was added to ensure the same reaction times of chromogen withthe enzyme in each well. Measure absorbance (AB) at 450 nm with areference wavelength of 630 nm.

Exemplary values. Exemplary values for the Standard Curve. The exemplaryvalues for the standards applies to those values at or above thequantitative limit (QL), as values below the QL are used only to helpestablish the extremes of the curve. The coefficient of determination(R²) for the standard curve should be ≥0.99, as determined by theSoftMax PRO software. The mean background for the zero ng/mL standardshould be ≤0.08 absorption units. The mean of the calculated values(ng/mL) at each standard concentration used to determine the standardcurve except zero and the QL must be within 15% of the target (nominal)value, as determined by the software. The coefficient of variation (%CV) of the triplicate AB₄₅₀ values at each standard concentration usedto determine the standard curve, excluding zero and QL must be ≤15%, asdetermined by the software. The mean of the triplicate absorbance valuesof the QL of the standard curve must exhibit a % CV of less than 20% andbe within 20% of target, as determined by the software. To ensure thatat lease two congruent data points are available for calculation, thestandards, controls and samples are loaded in triplicate wells. Eachvalue of the triplicate used to calculate the mean will be analyzedseparately.

-   -   For example:

Target Value (ng/mL) Actual Value (ng/mL) 2.5 1.2 2.3 2.5

The single value that is furthest from the target value of 2.5 ng/mL is1.2 ng/mL. By eliminating the 1.2 ng/mL value from the triplicate, themean of the remaining values meet all of the exemplary values. If, uponre-calculation, another point does not meet the exemplary values, thenthe assay is not valid and must be redone. If it is shown that the meanof the remaining two values still do not meet the exemplary values, thenthe single point (all 3 wells) is eliminated and the curve isre-calculated.

Exemplary values for QC Samples. A QC sample is defined as a set ofthree wells at the stated concentration, therefore, for the threenominal concentrations stated in this method there are a total of six QCsamples (for a total of 18 wells). At least two of the three wells for aQC sample must be within 20% of the nominal for the QC sample to beacceptable. At least four of the six QC samples must be acceptable; twoof the six QC samples (not two at the same concentration) and not morethan 6 of the 18 QC sample wells may deviate more than 20% of therespective target concentrations.

Exemplary values for Test Samples. Each of the values of the triplicateof the test sample assayed must be within 20% of the mean value at thatconcentration, as determined by the software. If two or more mean AB₄₅₀values cannot be calculated because they lie above the highest point onthe standard curve, then the sample must be re-assayed at higherdilutions until at least two of the three values fall on the standardcurve. The % CV of the triplicate observations obtained for each testsample target concentration must be ≤20%, as determined by the software.

Calculations. Refer to the SoftMax program template for the Protein AELISA as it generates mean, standard deviations and % CV. Average thetriplicate Absorbance (AB) values obtained for each reference and sampleconcentration assayed. Model the data for the Protein A standards usingan unweighted four parameter regression of the form:

${AB} = \frac{\min - \max}{1 + \left( \frac{C}{{ED}_{50}} \right)^{B}}$

-   -   Where:    -   min=AB value corresponding to the minimal asymptote    -   max=AB value corresponding to the maximal asymptote    -   ED₅₀=AB corresponding to one half the absolute difference        between the maximal and minimal asymptotic values    -   B=the approximated slope of the linear portion of the curve    -   C=Concentration of Protein A

Calculation of Protein A concentration in samples. Multiply the meansample results by the appropriate dilution factor (i.e. 2, 4, and 8), toobtain the concentration of Protein A in the original sample in ng/mL.Divide the results by the reported CTLA4-Ig protein concentration(mg/mL) to obtain the concentration of Protein A, in ng/mg, in CTLA4-Ig.

Example Calculation:

$\frac{{Mean}\mspace{14mu} {Sample}\mspace{14mu} {Result}}{{Abatacept}\mspace{11mu} {Concentration}} = {\frac{235\mspace{14mu} {ng}\mspace{14mu} {Protein}\mspace{14mu} A\text{/}{mL}}{50\mspace{14mu} {mg}\text{/}{mL}} = {4.7\mspace{14mu} {ng}\text{/}{mg}}}$Note:  ng  Protein  A/mg  CTLA 4-Ig  (ng/mg)  is  equivalentto  parts  per  million  (ppm).

Calculations for the Concentration of Protein A in Ng/mL of Samples. Theamount of Protein A in the test samples is calculated using currentSoftMax software using the regression equation. Three dilutions of eachsample are assayed. The calculated concentration is multiplied by theappropriate dilution factor to give the concentration in the initialtest sample.

-   -   Example:    -   The concentration of the test sample is 50 mg/mL.

Sample Dilution Dilution Factor   5 mg/mL 10  2.5 mg/mL 20 1.25 mg/mL 40

-   -   5 mg/mL sample-ELISA data determines concentration of Protein A        is 10 ng/mL    -   10 ng/mL×10 (dilution factor)=100 ng/mL Protein A in the test        sample    -   2.5 mg/mL sample-ELISA data determines concentration of Protein        A is 5.0 ng/mL    -   5 ng/mL×20 (dilution factor)=100 ng/mL Protein A in the test        sample    -   1.25 mg/mL sample-ELISA data determines concentration of Protein        A is 2.5 ng/mL    -   2.5 ng/mL×40 (dilution factor)=100 ng/mL Protein A in the test        sample

Calculate the mean concentration from the three values.

Reporting of Results. Results may be reported in terms of “% w/w TotalProtein, ng Protein A/mg Total Protein”, otherwise referred to as “ppm”(w/w).

-   -   Example:

0.0001%  w/w = 1  ng/mg = 1  ppm  (w/w)Example  Calculation:${\frac{0.0001\mspace{14mu} {mg}\text{/}{mL}\mspace{14mu} {Protein}\mspace{14mu} A}{50\mspace{14mu} {mg}\text{/}{mL}\mspace{14mu} {Test}\mspace{14mu} {Sample}} \times 100\%} = {{2\mspace{14mu} {ng}\text{/}{mg}} = {2\mspace{14mu} {ppm}}}$

Samples which result in an AB₄₅₀ value smaller than the AB₄₅₀ of QL(0.188 ng/mL) should be reported as “less than QL.” Samples which resultin an AB₄₅₀ value larger than the AB₄₅₀ of QL (0.188 ng/mL) should bereported to the nearest whole number as parts per million (ppm).

-   -   Example:    -   Sample concentration is 50 mg/mL. The highest sample        concentration analyzed is 5 mg/mL (1/10 dilution). The AB₄₅₀ of        the sample is smaller than QL.    -   QL=0.188 ng/mL    -   ≤0.188 ng/5 mg    -   ≤0.04 ng/mg    -   Report as “≤, QL=0.04 ppm”

Example 63 Methods to Obtain Molar Ratios of Amino Monosaccarides(N-Acetyl Galactosamine, N-Acetyl Glucosamine) to Protein in CTLA4-IgDrug Substance Samples

Reagents

Hydrolysis Solution (4 N HCl aqueous solution). Add 160 mL of 6 N HCland 80 mL of HPLC grade water to a 250 mL glass bottle. Stir to mixwell. Store at 2-8° C. for up to 6 months. Derivatization Solution I(0.1 M APTS aqueous solution). Add 192 μL of HPLC grade water to 10 mgpowder of APTS in a glass vial. Vortex the vial 5-10 seconds tocompletely dissolve the APTS. Store at −20° C. for up to one year.

Derivatization Solution II (1 M acetic acid and 0.25 M NaBH₃CN). Dilute20 μL acetic acid with 320 μL HPLC grade water (17 fold dilution) in a0.4 mL centrifuge tube to make a 1 M acetic acid solution. Weigh 2.0±0.5mg of NaBH₃CN into a cryogenic vial. Using the following formula, add anappropriate volume of the 1 M acetic acid solution to make 0.25 MNaBH₃CN. Volume (pL)=10³×(weight of NaBH₃CN in mg)/(62.84 g/mol×0.25mol/L). Note: Prepare immediately before use. Sodium cyanoborohydride(NaBH₃CN) should be stored in dark in a desiccator. Subdividing of thereagent into a series of 2.0 mL cryovials for storage is recommended toavoid repeated opening of the original reagent bottle as follows: Weigh1.0 g±0.2 mg of Sodium Cyanoborohydride into 2.0 mL cryovial. Aliquotout the entire contents of Sodium Cyanoborohydride from the originalbottle in this manner. Cap tightly and label cryovials sequentially(1,2,3, etc.) along with reagent name, lot number, and a 6 monthexpiration date. The vials should be sealed with parafilm to avoidmoisture. Weigh out Sodium Cyanoborohydride for Derivatization SolutionII no more than three times from the same cryovial. Make note of thisand the cryovial sequence number on the lab worksheet. Either a reagentpeak observed in the CE profile or poor labeling may occur afterrepeated opening of the cryovial or with that particular lot of SodiumCyanoborohydride. If this effects the results, discard the cryovialbeing used and either weigh out reagent from a cryovial with the nextsequence number or from a new lot of Sodium Cyanoborohydride.

Re-acetylation Buffer (25 mM sodium bicarbonate, pH 9.5). Weigh0.210±0.02 g of sodium bicarbonate into a clean 100 mL clean glassbeaker. Add 90 mL of HPLC grade water, and mix on a stir plate untilsalts are completely dissolved. Adjust the pH to 9.5±0.1 with 10 N NaOH.Add HPLC grade water to make the final volume 100 mL. Filter thesolution and store at room temperature for up to 3 months. RunningBuffer (60±5 mM Sodium tetraborate, pH 9.25). Weigh 1.21±0.02 g sodiumtetraborate into a 100 mL clean glass beaker. Add 90 mL of HPLC gradewater, and mix on a stir plate until salts are completely dissolved.Adjust the pH to 9.25±0.10 with 10 N NaOH. Add HPLC grade water to makethe final volume 100 mL for a final concentration of 60±5 mM. For a 55mM solution, weigh 1.11 g (±0.02) sodium tetraborate and follow aboveinstructions for dissolving and titrating. For a 65 mM solution, weigh1.31 g (±0.02) sodium tetraborate and follow above instructions fordissolving and titrating. Store at room temperature for up to 3 months.Prepare fresh buffer if peak resolution (as defined in systemsuitability section) is effected (R value <1.0). Optional: Dilutetetraborate buffer solution (MicroSolv) by adding 120 mL of ultra purewater to 80 mL of 150 mM sodium tetraborate buffer for a finalconcentration of 60 mM (±5 mM). Titrate with 10N NaOH to bring thesolution pH to 9.25 (±0.1). For a 55 mM tetraborate solution, dilute 66mL of 150 mM sodium tetraborate buffer into 114 mL of ultra pure water.Titrate as above. For a 65 mM tetraborate solution, dilute 78 mL of 150mM sodium tetraborate buffer into 102 mL of ultra pure water. Titrate asabove. Store the solution at room temperature for a maximum of 3 months.Prepare fresh buffer if peak resolution (as defined in systemsuitability section) is effected (R value <1.0).

Capillary Rinsing Solutions.

1 N NaOH solution: Add 1 mL of 10 N NaOH solution to a 15 mL graduatedplastic tube containing 9 mL of HPLC grade water. Mix well by vortexing5-10 sec. Store the solution at room temperature for up to 6 months.

1 N HCl solution: Add 1 mL of 6 N HCl solution to a 15 mL graduatedplastic tube containing 5 mL of HPLC grade water. Mix well by vortexing5-10 sec. Store the solution at room temperature for up to 6 months. 80%methanol solution: Add 8 mL HPLC grade methanol to a 15 mL graduatedplastic tube containing 2 mL HPLC grade water. Mix well by vortexing5-10 sec. Store the solution at room temperature for up to 6 months.

Monosaccharide Standard Stock Solutions:

N-Acetyl Glucosamine (GalNAc). Accurately weigh 5±1 mg of GalNAc into a2.0 mL cryogenic vial. Add 1 mL of HPLC grade water and mix well byvortexing until dissolved. Record the accurate concentration of thesolution (mg/mL).

N-Acetyl Galactosamine (GlcNAc): Accurately weigh 5±1 mg of GlcNAc intoa 2.0 mL cryogenic vial. Add 1 mL of HPLC grade water and mix well byvortexing until dissolved. Record the accurate concentration of thesolution (mg/mL).

N-Acetyl Mannosamine (ManNAc): Accurately weigh 5±1 mg of ManNAc into a2.0 mL cryogenic vial. Add 1 mL of HPLC grade water and mix well byvortexing until dissolved. Record the accurate concentration of thesolution (mg/mL).

Store Monosaccharide Standard Stock Solutions at −20° C. for up to 1year:

Monosaccharide Working Solution I: Internal Standard Working Solution.Dilute stock solution of ManNAc 100 fold with HPLC grade water by adding20 μL of ManNAc stock solution into a 2 mL cryogenic vial which alreadycontains 1980 μL of HPLC grade water. Vortex approximately 5 to 10seconds. Store the internal standard working solution at 2-8° C. for upto 6 months.

Monosaccharide Working Solution II: Amino Mix Standard Working Solution.In a 2.0 mL cryogenic vial containing 1960 μL of HPLC grade water, add20 μL of stock solutions of GalNAc and GlcNAc, respectively. Vortexapproximately 5 to 10 seconds. Store the amino mix standard workingsolution at 2-8° C. for up to 6 months.

Sample and reference material solutions. Thaw frozen protein samples at2-8° C., and gently mix by inversion. Dilute both samples and referencematerial with HPLC grade water to about 1.0 mg/mL. Make note ofconcentration out to three significant figures.

CE Running Conditions.

Running Buffer (step 2.5) 60 mM sodium tetraborate, pH 9.25 CapillaryCartridge temperature 25° C. Voltage 25-30 kV, positive mode Detectorcondition LIF detector, Excitation: 488 nm, Emission: 520 nm. Sampleinjection Pressure injection mode, 20 s at 0.5 PSI Run Time 10 minutesSample storage 10° C.

Procedure

Hydrolysis. In a 0.65 mL centrifuge tube, add 10 μL of ManNAc workingsolution and 200 μL 4 N Hydrolysis Solution. This serves as a systemblank. In a 0.65 mL centrifuge tube, add 10 μL of ManNAc workingsolution and 10 μL of Amino Mix Standard Solution. Further add 200 μL of4N Hydrolysis Solution. This serves as monosaccharide standard forquantitation and System Suitability. Prepare in duplicate. In a 0.65 mLcentrifuge tube, add 10 μL of ManNAc working solution and 10 μL ofCTLA4-Ig reference material solution (approximately 1 mg/mL). Furtheradd 200 μL of 4N HCl solution. Prepare in duplicate. In a 0.65 mLcentrifuge tube, add 10 μL of ManNAc working solution and 10 μL ofsample solution (approximately 1 mg/mL). Further add 200 μL of 4N HClsolution. Prepare in duplicate. Vortex samples for approximately 10seconds and centrifuge for approximately 5-10 seconds. Place samples ina 96-position vial rack and incubate in an oven at 95° C. for 6 hr.After hydrolysis, place hydrolyzed samples at −20° C. for 10 minutes tocool down. Briefly centrifuge the hydrolyzed samples until anycondensate is forced to the bottom of the tube (5-10 seconds at highspeed). Evaporate samples to dryness in SpeedVac. Reconstitute eachsample with 100 μL of HPLC grade water and vortex 10-15 sec. Evaporatesamples to dryness in SpeedVac.

Re-acetylation. Reconstitute each sample with 10 μL of M6 re-acetylationbuffer and vortex 5-10 sec. to mix well. Add 4 μL of M3 re-acetylationreagent into each tube. Vortex for approximately 5-10 seconds. Incubateon ice for 30 minutes. Evaporate samples to dryness in SpeedVac.Reconstitute each sample with 100 μL of HPLC grade water and vortex10-15 sec. Evaporate samples to dryness in SpeedVac.

Derivatization. Place the micro centrifuge in the oven to equilibrate tothe oven temperature of 55° C. Reconstitute each sample with 10 μL ofDerivitization Solution I (0.1 M APTS solution). Vortex approximately5-10 seconds. Add 5 μL of the Derivatization Solution II (1M HAc and0.25 M NaBH₃CN). Vortex approximately 5-10 seconds and centrifuge.Quickly load the sample vials into the pre-warmed centrifuge, and placethe centrifuge back in the 55° C. oven. Incubate for 3 hr whilecentrifuging at 2000 rpm. This prevents the condensation of solvent onvial surface.

Instrumentation Preparation.

5.4.1 Installing a new capillary, rinse in high pressure mode (80 PSI)using the following steps: 1 N NaOH for 20 minutes.; HPLC grade waterfor 10 minutes. 60 mM sodium tetraborate buffer for 10 minutes.

Daily Operation

Before each day's operation, run the washing/rinse sequences to rinsethe capillary.

Then run the System Suitability Standard (monosaccharide standard) toensure the system is suitable.

Using 1N NaOH may etch the inside of capillaries from different vendorsand cause a shift in migration times throughout the run. If this causesthe migration time of the last peak (G1cNAc) to be more than 10.0minutes, it may be necessary to replace 1N NaOH with 0.1N NaOH or HPLCgrade water for the step 2 rinse.

When using an equivalent capillary and the above washing procedure isnot adequate using 80% methanol and/or 1N HCl may be necessary for thelast peak (G1cNAc) to be within the exemplary values of 10.0 minutes.

Preparation for Injection

After derivatization, let samples cool down to room temperature.Centrifuge approximately 10 seconds at room temperature, untilcondensate is forced to the bottom of the tube.

Add 85 μL of HPLC grade water to each tube to bring the final volume ofeach sample to 100 pt. Vortex for 5-10 seconds.

Transfer 10pL of sample from each tube to a CE micro vial and add 190 μLof HPLC grade water to each tube. Vortex for 5-10 seconds.

Rinse steps and Injection sequence:

-   -   Note: For every four injections, change the CE running buffer        with newly prepared CE running buffer (due to ionic depletion        effect). Perform capillary rinse at 40 psi.

Run Time Step Description (min)  1 (Rinse) HPLC grade water 1  2 (Rinse)1N NaOH or 0.1N NaOH 3 OR HPLC grade water 1 Note: When using HPLC waterfor the step 2 rinse, steps 1, 2, and 3 may be combined in a single 3minute run.  3 (Rinse) HPLC grade water 1  4 (Rinse) 60 mM sodiumTetraborate Run Buffer 5  5 (Rinse) Blank (Internal Standard Marker) 10 6 (Rinse) Repeat 1-4 10  7 (Rinse) System Suitability (Mono Std prep1)10  8 (Rinse) Repeat 1-4 10  9 System Suitability (Mono Std prep 1) 1010 (Rinse) Repeat 1-4 10 11 System Suitability (Mono Std prep 2) 10 12(Rinse) Repeat 1-4 10 13 System Suitability (Mono Std prep 2) 10 14(Rinse) Repeat 1-4 10 15 CTLA4-Ig Ref. Mat. prep 1 10 16 (Rinse) Repeat1-4 10 17 CTLA4-Ig Ref. Mat. prep 2 10 18 (Rinse) Repeat 1-4 10 19Sample 1 prep 1 10 20 (Rinse) Repeat 1-4 10 21 Sample 1 prep 1 10 22(Rinse) Repeat 1-4 10 23 Sample 1 prep 2 10 24 (Rinse) Repeat 1-4 10 25Sample1 prep 2 10 26 (Rinse) Repeat 1-4 18 27 Sample 2 prep1 10 28(Rinse) Repeat 1-4 10 29 Sample 2 prep 1 10 30 (Rinse) Repeat 1-4 10 31Sample 2 prep 2 10 32 (Rinse) Repeat 1-4 10 33 Sample 2 prep 2 10 34(Rinse) Repeat 1-4 10 35 Sample 3 prep 1 15 36 (Rinse) Repeat 1-4 10 37Sample 3 prep 1 10 38 (Rinse) Repeat 1-4 10 39 Sample 3 prep 2 10 40(Rinse) Repeat 1-4 10 41 Sample 3 prep 2 10 42 (Rinse) Repeat 1-4 10 43CTLA4-Ig Reference Material prep 1 10 44 (Rinse) Repeat 1-4 10 45CTLA4-Ig Reference Material prep 2 10 46 (Rinse) Repeat 1-4 10 47 SystemSuitability (Mono Std prep 1) 10 48 (Rinse) Repeat 1-4 10 49 SystemSuitability (Mono Std prep 1) 10 50 Repeat 1-4 10 51 System Suitability(Mono Std prep 2) 10 52 Repeat 1-4 10 53 System Suitability (Mono Stdprep 2) 10 *Note: Repeat sequence for up to three samples in duplicateand bracket with 2 injections of each preparation of Monosaccharidestandard. Use all eight System Suitability Standard injections forsamples placed in groups of three. If running more than three samples,run the additional samples as shown in the above sequence beginning withline 19. Complete the sequence by running the System Suitability (MonoStd) as shown in lines 47 thru 53 in Table **Bracket samples with twoinjections of each preparation of CTLA4-Ig reference material.

System Suitability Note: System suitability values are determined usingthe first injection of system suitability standard unless otherwisespecified. The electropherogram of the first system suitability shouldbe similar to that shown in FIG. 1, where peak 1 is GalNAc; peak 2 isManNAc; peak 3 is GlcNAc. Note: When CE instruments other than BeckmanPACE MDO are to be used, the length of the capillary might be differentfrom that specified in this method due to various configurations ofcartridges holding the separation capillary. This would cause variationsin analyte migration time, as well as peak intensity.

Resolution between two neighbor peaks is calculated for the first SystemSuitability standard by the instrument according to the followingequation:

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{\left( {W_{1} + W_{2}} \right)}$

-   -   Where:    -   R=resolution    -   t₂, t₁=migration times of the two neighbor peaks respectively    -   W₁, W₂=peak widths at baseline of the two neighbor peaks        respectively    -   The R value must be ≥1.0. If R <1.0, rinse the capillary with        the washing/rinse sequences; if the problem persists, replace        old buffer with freshly prepared Running Buffer or replace the        capillary.

For the last System Suitability injection, the last peak (GlcNAc) musthave a tailing factor <1.4 using the following formula:

T=W_(0.05)/2f

-   -   Where:    -   T=tailing factor    -   W_(0.05)=width of peak at 5% of height    -   f=width of the peak front at peak maximum    -   If T ≥1.4, rinse the capillary with the washing/rinse sequences;        if the problem persists, replace old buffer with freshly        prepared run buffer or replace the capillary.    -   6.1 The replicate injections of System Suitability Standards        must meet the following exemplary values:    -   Peak Area Ratio of GlcNAc vs. MaNAc: RSD ≤10% (calculated in        step 7.1)    -   Migration time of GlcNAc should be ≤10.0 minutes    -   Profile should be equivalent to FIG. 1 where the three peaks are        observed and the

Internal Standard (ManNAc) is the number 2 peak.

If any of the above exemplary values are not met prior to testingsamples, first increase the voltage if the migration time of GlcNAc isgreater than 10.0 minutes. Next, if the peak area ratio is >10%, preparefresh CE buffer making certain of its pH or replace the capillary. Afteradjustment to the instrument, repeat System Suitability injections. Whenanalyzing the peak profile, if a significant decrease in the peak heightof ManNac occurs, check to make certain the fiber optic cable into theLIF module is not misaligned.

Determine monosaccharide standard percent RSD by comparing peak arearatios of internal standard and monosaccharide standard components.Divide the peak area for each monosaccharide component by the peak areaof the internal standard for each monosaccharide standard injection.Calculate the percent RSD for GalNAc and GlcNAc for the two, bracketedstandards. The RSD should be ≤10%. If this averaging exemplary value isnot met, then the capillary should be rinsed or replaced as above.

Calculations

Calculating Peak Area Ratio of GalNAc and GlcNAc relative to theInternal Standard (ManNAc). Used on replicate injections of first fourSystem Suitability Standards so as to meet exemplary values andperforming same calculations on all of the bracketed, System SuitabilityStandards injected before and after sample(s).

Peak Area Ratio=Divide the peak area for each monosaccharide component(GlcNAc, GalNAc) by the peak area of the internal standard (ManNAc) foreach System Suitability Standard injection.

${{Peak}\mspace{14mu} {Area}\mspace{14mu} {Ratio}} = \frac{{monosaccharide}\mspace{14mu} {peak}\mspace{14mu} {area}}{{MaNAc}\mspace{14mu} {peak}\mspace{14mu} {area}}$

Calculate a mean of the Peak Area Ratios for GlcNAc and GalNAc in theSystem Suitability Standards. Also calculate a Standard Deviation (S.D.)and percent relative standard deviation (% RSD)

Exemplary values: RSD for the Peak Area Ratio of GlcNAc ≤10%. Two,bracketed, System Suitability Standards injected before and aftersample(s): Percent RSD for the Peak Area Ratio of GlcNAc and GalNAc≤10%.

If this averaging exemplary value is not met (RSD >10%), then thecapillary needs to be re-rinsed with the rinse procedures and thosesamples and bracketed monosaccharide standards need to be run again. Ifthe averaging exemplary value is still not met, replace the capillaryand rinse as stated. Run the samples and bracketed monosaccharidestandards again.

${{Standard}\mspace{14mu} {Deviation}} = \sqrt{\frac{{n\; \Sigma \; x^{2}} - \left( {\Sigma \; x} \right)^{2}}{n\left( {n - 1} \right)}}$

-   -   Where:    -   n=number of measurements in the sample    -   x=individual measurements

${\% \mspace{14mu} {RSD}} = {\frac{{Standard}\mspace{14mu} {Deviation}}{{Average}\mspace{14mu} {Measured}\mspace{14mu} {Peak}\mspace{14mu} {Area}} \times 100}$

Calculate the molar ratio of GalNAc/Protein:

$R_{GalNAc} = \frac{A_{GalNAc} \times A_{{ManNAc}\; 0} \times V_{{GalNAc}\; 0} \times C_{{GalNAc}\; 0} \times {MW}_{{CTLA}\; 4\text{-}{Ig}}}{A_{ManNAc} \times A_{{GalNAc}\; 0} \times {Vp} \times {Cp} \times {MW}_{GlcNAc}}$

-   -   Where:    -   R_(GalNAc)=molar ratio of GalNAc vs. protein    -   A_(GalNAc)=peak area (μV·sec) of GalNAc in sample    -   A_(ManNAc)=peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0)=peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(GalNAc0)=peak area (μV·sec) average of GalNAc in        monosaccharide standard    -   V_(GalNAc0)=volume of GalNAc contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(GalNAc0)=concentration of GalNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig Reference Material    -   MW_(GlcNAc)=Molecular weight of GalNAc (221.2 daltons)

Standards Bracketing. When calculating molar ratios of CTLA4-Igreference material and samples, use all eight of the bracketed SystemSuitability Standards. Average the peak areas for inclusion in thisequation. This is to be used for the first three samples. For all othersamples, always use the average peak area of the next four bracketedmonosaccharide standards and the previous four bracketed monosaccharidestandards for molar ratio calculations.

Calculate the molar ratio of GlcNAc/Protein

$R_{GalNAc} = \frac{A_{GalNAc} \times A_{{ManNAc}\; 0} \times V_{{GalNAc}\; 0} \times C_{{GalNAc}\; 0} \times {MW}_{{CTLA}\; 4\text{-}{Ig}}}{A_{ManNAc} \times A_{{GalNAc}\; 0} \times {Vp} \times {Cp} \times {MW}_{GlcNAc}}$

-   -   Where:    -   R_(GlcNAc)=molar ratio of GlcNAc vs. protein    -   A_(GlcNAc)=peak area (μV·sec) of GlcNAc in sample    -   A_(ManNAc)=peak area (μV·sec) of ManNAc in sample    -   A_(ManNAc0)=peak area (μV·sec) average of ManNAc in        monosaccharide standard    -   A_(G1cNAc0)=peak area (μV·sec) average of GlcNAc in        monosaccharide standard    -   V_(GlcNAc0)=volume of GlcNAc contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(GlcNAc0)=concentration of GlcNAc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig Reference Material as        per COA    -   MW_(GlcNAc)=Molecular weight of GlcNAc (221.2 daltons)

Exemplary values. The percent RSD for the two, bracketed, amino SystemSuitability Standard peak area ratios should not exceed 10%. The averagemolar ratios for amino monosaccharides in the reference material must bewithin the ranges specified. For each component, the % RSD for the fourresults (duplicate injection of duplicate preparations) must be </=25%.

Molar Ratio range of CTLA4-Ig Reference Material Monosaccharide RangeGalNAc 2.0-3.2 GlcNAc 18-32

Reporting Results. Report the average result as the number of GalNAcmolecules per CTLA4-Ig molecule and number of GlcNAc molecules perCTLA4-Ig molecule. Report molar ratio results to two significantfigures. For each component, the % RSD for the four results (duplicateinjection of duplicate preparations) must be </=25%.

Example 64 Methods to Obtain Molar Ratios of Neutral Monosaccharides(Mannose, Fucose, Galactose) to Protein in CTLA4-Ig Drug SubstanceSamples

Reagents

Hydrolysis Solution (2 M TFA aqueous solution) Add 148 μL of TFA and 852μL of HPLC grade water to a 1.7 microcentrifuge tube. Vortex for 5-10seconds. Scale up as needed. Prepare solution immediately before use.

Derivatization Solution I (0.1 M APTS aqueous solution). Add 192 μL ofHPLC grade water to 10 mg powder of APTS in a glass vial. Vortex thebottle 5-10 seconds to completely dissolve the APTS. Store at −20° C.for up to one year.

Derivatization Solution II (1 M acetic acid and 0.25 M NaBH₃CN). Dilute20 μL acetic acid with 320 μL HPLC grade water (17 fold dilution) in a0.4 mL centrifuge tube to make a 1 M acetic acid solution. Weigh 2.0±0.5mg of NaBH₃CN into a cryogenic vial. Using the following formula, add anappropriate volume of the 1 M acetic acid solution to make 0.25 MNaBH₃CN.

Volume(μL)=10³×(weight of NaBH₃CN in mg)/62.84 g/mol×0.25 mol/L)

-   -   Sodium cyanoborohydride (NaBH₃CN) should be stored in dark in a        desiccator.    -   Subdividing of the reagent into a series of 2.0 mL cryovials for        storage is recommended to avoid repeated opening of the original        reagent bottle as follows:    -   Weigh 1 g±0.2 mg of Sodium Cyanoborohydride into 2.0 mL        cryovial. Aliquot out the entire contents of Sodium        Cyanoborohydride from the original bottle in this manner.    -   Cap tightly and label cryovials sequentially (1,2,3, etc.) along        with reagent name, lot number, and a 6 month expiration date.    -   The vials should be sealed with parafilm to avoid moisture.    -   Weigh out Sodium Cyanoborohydride for Derivatization Solution II        no more than three times from the same cryovial. Make note of        this and the cryovial sequence number on the lab worksheet.    -   Either a reagent peak observed in the CE profile or poor        labeling may occur after repeated opening of the cryovial or        with that particular lot of Sodium Cyanoborohydride. If this        effects the results, discard the cryovial being used and either        weigh out reagent from a cryovial with the next sequence number        or from a new lot of Sodium Cyanoborohydride.

Running Buffer (60±5 mM Sodium tetraborate, pH 9.25)

-   Weigh 1.21±0.02 g sodium tetraborate into a 100 mL clean, glass    bottle.-   Add 90 mL of HPLC grade water, and mix on a stir plate until salts    are completely dissolved.-   Adjust the pH to 9.25±0.10 with 10 N NaOH.-   Add HPLC grade water to make the final volume 100 mL for a final    concentration of 60 mM (±5 mM).-   For a 55 mM solution, weigh 1.11 g (±0.02) sodium tetraborate and    follow above instructions for dissolving and titrating.-   For a 65 mM solution, weigh 1.31 g (±0.02 g) sodium tetraborate and    follow above instructions for dissolving and titrating.-   Store at room temperature for up to 3 months. Prepare fresh buffer    if peak resolution (as defined in system suitability section) is    effected (R value <1.0.-   Optional: Dilute tetraborate buffer solution (MicroSolv) by adding    120 mL of ultra pure water to 80 mL of 150 mM sodium tetraborate    buffer for a final concentration of 60 mM (±5 mM). Titrate with 10N    NaOH to bring the solution pH to 9.25 (±0.1).-   For a 55 mM tetraborate solution, dilute 66 mL of 150 mM sodium    tetraborate buffer into 114 mL of ultra pure water. Titrate as    above.-   For a 65 mM tetraborate solution, dilute 78 mL of 150 mM sodium    tetraborate buffer into 102 mL of ultra pure water. Titrate as    above.-   Store the solution at room temperature for a maximum of 3 months.    Prepare fresh buffer if peak resolution (as defined in system    suitability section) is effected (R value <1.0.

Capillary Rinsing Solutions

1 N NaOH solution

-   -   Add 1 mL of 10 N NaOH solution to a 14 mL graduated plastic tube        containing 9 mL of HPLC grade water. Mix well by vortexing 5-10        sec.        -   Store the solution at room temperature for up to 6 months.

1 N HCl solution:

-   -   Add 1 mL of 6 N HCl solution to a 15 mL graduated plastic tube        containing 5 mL of HPLC grade water. Mix well by vortexing 5-10        sec.        -   Store the solution at room temperature for up to 6 months.

80% methanol solution:

-   -   Add 8 mL HPLC grade methanol to a 15 mL graduated plastic tube        containing 2 mL HPLC grade water. Mix well by vortexing 5-10        sec.    -   Store the solution at room temperature for up to 6 months.

Monosaccharide standard stock solutions

Mannose (Man)

-   -   Accurately weigh 5±1 mg of mannose into a 2.0 mL cryogenic vial.    -   Add 1 mL of HPLC grade water and mix well by vortexing 5-10 sec.    -   Record the accurate concentration of the solution (mg/mL).

Fucose (Fuc)

-   -   Accurately weigh 5±1 mg of fucose into a 2.0 mL cryogenic vial.    -   Add 1 mL of HPLC grade water and mix well by vortexing 5-10 sec.    -   Record the accurate concentration of the solution (mg/mL).

Galactose (Gal)

-   -   Accurately weigh 5±1 mg of galactose into a 2.0 mL cryogenic        vial.    -   Add 1 mL of HPLC grade water and mix well by vortexing 5-10 sec.    -   Record the accurate concentration of the solution (mg/mL).

Xylose (Xyl)

-   -   Accurately weigh 5±1 mg of xylose into a 2.0 mL.    -   Add 1 mL of HPLC grade water and mix well by vortexing 5-10 sec.    -   Record the accurate concentration of the solution (mg/mL).

Store the monosaccharide standard stock solutions at −20° C. for up to 1year.

Monosaccharide working solution I: Internal standard working solution.To make internal standard working solution, dilute stock solution ofxylose 100 times with HPLC grade water by adding 20 μL of xylose stocksolution (3.6.4) into a 2 mL cryogenic vial, which already contains 1980μL of HPLC grade water. Vortex for approximately 5 to 10 seconds. Storethis internal standard working solution at 2-8° C. for up to 6 months.

Monosaccharide working solution II: Neutral mix standard workingsolution. In a 2.0 mL cryogenic vial containing 1940 μL of HPLC gradewater, add 20 μL of stock solutions of mannose, fucose, and galactoserespectively. Vortex for approximately 5 to 10 seconds. Store thisinternal standard working solution at 2-8° C. for up to 6 months.

Sample and reference material solutions. Thaw frozen protein samples at2-8° C. and gently mix by inversion. Dilute both samples and referencematerial with HPLC grade to about 1.0 mg/mL.

CE Running Conditions

Running Buffer 60 mM sodium tetraborate, pH 9.25 Capillary Cartridgetemperature 25° C. Voltage 25-30 kV, positive mode Detector conditionLIF detector Excitation: 488 nm, Emission: 520 m Sample injectionPressure injection mode, 20 s at 0.5 PSI Run Time 15 minutes Samplestorage 10° C.

Procedure

Hydrolysis: In a 0.65 mL centrifuge tube, add 10 μL of xylose workingsolution and 200 μL 2M TFA solution. This serves as a system blank. In a0.65 mL centrifuge tube, add 10 μL of xylose working solution and 10 μLof neutral mix standard working solution. Further add 200 μL of 2M TFAsolution and vortex for 3-4 sec. This serves as monosaccharide standardfor quantitation and System Suitability. Prepare in duplicate. In a 0.65mL centrifuge tube, add 10 μL of xylose working solution and 10 μL ofCTLA4-Ig reference material solution (approximately 1 mg/mL). Furtheradd 200 μL of 2M TFA solution and vortex for 3-4 sec. Prepare induplicate. In a 0.65 mL centrifuge tube, add 10 μL of xylose workingsolution and 10 μL of sample solution (approximately 1 mg/mL). Furtheradd 200 μL of 2M TFA solution and vortex for 3-4 sec. Prepare induplicate. Vortex samples for approximately 20 seconds and centrifugefor approximately 5 to 10 seconds. Place samples in a 96-position vialrack and incubate in an oven at 95° C. for 6 hr. After hydrolysis, placesamples at −20° C. for 10 minutes to cool down. Briefly centrifugehydrolyzed samples until any condensate is forced to the bottom of thetube (5 to 10 seconds at high speed). Evaporate samples to dryness inSpeedVac. Reconstitute each sample with 100 μL of HPLC grade water andvortex 10-15 sec. Evaporate samples to dryness in SpeedVac.

Derivatization: Place the micro centrifuge in the oven to equilibrate tothe oven temperature of 55° C. Reconstitute each sample with 10 μL ofDerivatization Solution I (0.1 M APTS solution). Vortex approximately5-10 seconds. Add 5 μL of the Derivatization Solution II (1M HAc and0.25 M NaBH₃CN). Vortex approximately 5-10 seconds and centrifuge.Quickly load the sample vials into the pre-warmed centrifuge, and placethe centrifuge back in the 55° C. oven. Incubate for 3 hr whilecentrifuging at 2000 rpm. This prevents the condensation of solvent onvial surface.

Instrumentation Preparation: Installing a new capillary, rinse in highpressure mode (80 PSI) using the following steps:

-   -   1 N NaOH for 20 minutes.    -   HPLC grade water for 10 minutes.    -   60 mM sodium tetraborate buffer for 10 minutes.

Run the washing/rinse sequences to rinse the capillary. Then run theSystem Suitability Standard (monosaccharide standard) to ensure thesystem is suitable. Using 1N NaOH may etch the inside of capillariesfrom different vendors and cause a shift in migration times throughoutthe run. If this causes the migration time of the last peak (galactose)to be more than 15.0 minutes, it may be necessary to replace 1N NaOHwith 0.1N NaOH or HPLC grade water for the step 2 rinse. When using anequivalent capillary and the above washing procedure is not adequateusing 80% methanol and/or 1N HCl may be necessary for the last peak(galactose) to be within the exemplary values of 15.0 minutes.

Preparation for injection: After derivatization, let samples cool downto room temperature. Centrifuge approximately 10 seconds at roomtemperature, until condensate is forced to the bottom of the tube. Add85 μL of HPLC grade water to each tube to bring the final volume of eachsample to 100 μL. Vortex for 5-10 seconds. Transfer 10pL of sample fromeach tube to a CE micro vial and add 190 μL of HPLC grade water to eachtube. Vortex for 5-10 seconds.

Rinse steps and Injection sequence:

Run Time Step Description (min)  1 (Rinse) HPLC grade water 1  2 (Rinse)1N NaOH or 0.1N NaOH 3 OR HPLC grade water 1 Note: When using HPLC waterfor the step 2 rinse, steps 1, 2, and 3 may be combined in a single 3minute run.  3 (Rinse) HPLC grade water 1  4 (Rinse) 60 mM sodiumTetraborate Run Buffer 5  5 Blank (Internal Standard Marker) 15  6(Rinse) Repeat 1-4 10  7 System Suitability (Mono Std prep 1) 15  8(Rinse) Repeat 1-4 10  9 System Suitability (Mono Std prep 1) 15 10(Rinse) Repeat 1-4 10 11 System Suitability (Mono Std prep 2) 15 12(Rinse) Repeat 1-4 10 13 System Suitability (Mono Std prep 2) 15 14(Rinse) Repeat 1-4 10 15 CTLA4-Ig ref. mat. prep 1 15 16 (Rinse) Repeat1-4 10 17 CTLA4-Ig Reference Material prep 2 15 18 (Rinse) Repeat 1-4 1019 Sample 1 prep 1 15 20 (Rinse) Repeat 1-4 10 21 Sample 1 prep 1 15 22(Rinse) Repeat 1-4 10 23 Sample 1 prep 2 15 24 (Rinse) Repeat 1-4 10 25Sample 1 prep2 15 26 (Rinse) Repeat 1-4 10 27 Sample 2 prep1 15 28(Rinse) Repeat 1-4 10 29 Sample 2 prep 1 15 30 Repeat 1-4 10 31 Sample 2prep 2 15 32 (Rinse) Repeat 1-4 10 33 Sample 2 prep 2 15 34 (Rinse)Repeat 1-4 10 35 Sample 3 prep 1 15 36 (Rinse) Repeat 1-4 10 37 Sample 3prep 1 15 38 (Rinse) Repeat 1-4 10 39 Sample 3 prep 2 15 40 (Rinse)Repeat 1-4 10 41 Sample 3 prep 2 15 42 (Rinse) Repeat 1-4 10 43 CTLA4-IgReference Material prep 1 15 44 (Rinse) Repeat 1-4 10 45 CTLA4-IgReference Material prep 2 15 46 (Rinse) Repeat 1-4 10 47 SystemSuitability (Mono Std prep 1) 15 48 (Rinse) Repeat 1-4 10 49 SystemSuitability (Mono Std prep 1) 15 50 Repeat 1-4 10 51 System Suitability(Mono Std prep 2) 15 52 Repeat 1-4 10 53 System Suitability (Mono Stdprep 2) 15 *Repeat sequence for up to three samples in duplicate andbracket with 2 injections of each preparation of Monosaccharidestandard. Use all eight System Suitability Standard injections forsamples placed in groups of three. If running more than three samples,run the additional samples as shown in the above sequence beginning withline 19. **Bracket samples with two injections of each preparation ofCTLA4-Ig reference material.

System Suitability. The electropherogram of the first system suitabilityshould be similar to where peak 1 is mannose; peak 2 is xylose; peak 3is fucose; and peak 4 is galactose. Note: When CE instruments other thanBeckman PACE MDQ are to be used, due to the various configuration ofcartridges holding the separation capillary, the length of the capillarymight be different from that specified in this method. This would causevariations in analyte migration time, as well as peak intensity.

Resolution between two neighbor peaks is calculated for the first SystemSuitability standard by the instrument according to the followingequation:

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{\left( {W_{1} + W_{2}} \right)}$

-   -   Where:    -   R=resolution    -   t₂, t₁=migration times of the two neighbor peaks respectively    -   W₁, W₂=peak widths at baseline of the two neighbor peaks        respectively    -   R value must be ≥1.0. If R <1.0, rinse the capillary with the        washing/rinse sequences; if the problem persists, replace old        buffer with freshly prepared run buffer or replace the        capillary.

For the last System Suitability injection, the last peak (galactose)must have a tailing factor <1.4 using the following formula:

T=W_(0.05)/2f

-   -   Where:    -   T=tailing factor    -   W_(0.05)=width of peak at 5% of height    -   f=width of the peak front at peak maximum    -   If T ≥1.4, rinse the capillary with the washing/rinse sequences;        if the problem persists, replace old buffer with freshly        prepared run buffer or replace the capillary.

Replicate injection of first four System Suitability Standards must meetthe following exemplary values:

-   -   Peak Area Ratio of galactose vs. xylose: RSD ≤10%.    -   Migration time of galactose needs to be ≤15.0 minutes    -   Profile should be equivalent to FIG. 1 where the four peaks are        observed and the Internal Standard (Xylose) is the number 2        peak.

If any of the above exemplary values are not met, first increase thevoltage if the migration time of galactose is greater than 15.0 minutes.Next, if the peak area ratio is >10%, then prepare fresh CE buffermaking certain of its pH or replace the capillary.

After adjustments to the instrument, repeat system suitabilityinjections. When analyzing the peak profile, if a significant decreasein the peak height of Xylose occurs, check to make certain the fiberoptic cable into the LIF module is not mis-aligned. Determinemonosaccharide standard percent RSD by comparing peak area ratios ofinternal standard and monosaccharide standard components. Divide thepeak area for each monosaccharide component by the peak area of theinternal standard for each monosacharide standard injection. Calculatethe percent RSD for mannose, fucose, and galactose for the two bracketedstandards. The RSD should be ≤10%. If this averaging exemplary value isnot met, then the capillary should be rinsed or replaced as above.Samples and bracketed monosaccharide standards need to be repeated.

Calculations. Calculating Peak Area Ratio of Man, Fuc, and Gal relativeto the Internal Standard (Xylose). Used on replicate injections of firstfour System Suitability Standards so as to meet exemplary values andperforming same calculations on all of the bracketed System SuitabilityStandards injected before and after sample(s). Peak Area Ratio=Dividethe peak area for each monosaccharide component (Man, Fuc, and Gal) bythe peak area of the internal standard (Xylose) for each SystemSuitability Standard injection.

${{Peak}\mspace{14mu} {Area}\mspace{14mu} {Ratio}} = \frac{{monosaccharide}\mspace{14mu} {peak}\mspace{14mu} {area}}{{Xylose}\mspace{14mu} {peak}\mspace{14mu} {area}}$

Calculate a mean of the Peak Area Ratios for Man, Fuc, and Gal in theSystem Suitability Standards. Also calculate a Standard Deviation (S.D.)and percent relative standard deviation (% RSD). Exemplary values: RSDfor the Peak Area Ratio of Galactose ≤10%. Two, bracketed, SystemSuitability Standards injected before and after sample(s):

Percent RSD for the Peak Area Ratio of Man, Fuc, and Gal ≤10%.

If this averaging exemplary value is not met (RSD >10%), then thecapillary needs to be re-rinsed with the rinse procedures and thosesamples and bracketed monosaccharide standards need to be run again. Ifthe averaging exemplary value is still not met, replace the capillaryand rinse. Run the samples and bracketed monosaccharide standards again.

${{Standard}\mspace{14mu} {Deviation}} = \sqrt{\frac{{n\; \Sigma \; x^{2}} - \left( {\Sigma \; x} \right)^{2}}{n\left( {n - 1} \right)}}$

-   -   Where:    -   n=number of measurements in the sample    -   x=individual measurements

${\% \mspace{14mu} {RSD}} = {\frac{{Standard}\mspace{14mu} {Deviation}}{{Average}\mspace{14mu} {Measured}\mspace{14mu} {Peak}\mspace{14mu} {Area}} \times 100\%}$

Calculate the molar ratio of Mannose/Protein

$R_{Man} = \frac{A_{Man} \times A_{{Xy}\; 10} \times V_{{Man}\; 0} \times C_{{Man}\; 0} \times {MW}_{{CTLA}\; 4\text{-}{Ig}}}{A_{{Xy}\; 1} \times A_{{Man}\; 0} \times V_{p} \times C_{p} \times {MW}_{Man}}$

-   -   Where:    -   R_(Man)=molar ratio of Mannose (Man) vs. protein    -   A_(Man)=peak area (μV·sec) of Man in sample    -   A_(Xyl)=peak area (μV·sec) of Xylose (Xyl) in sample    -   A_(Xyl0)=peak area (μV·sec) average of Xyl in monosaccharide        standard    -   A_(Man0)=peak area (μV·sec) average of Man in monosaccharide        standard    -   V_(Man0)=volume of Man contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(Man0)=concentration of Man contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig Reference Material as        per Certificate of Analysis (COA)    -   MW_(Man)=Molecular weight of Mannose (180.2 daltons)

Standard Bracketing. When calculating molar ratios of CTLA4-Ig referencematerial and samples, use all eight of the bracketed System SuitabilityStandards. Average the peak areas for inclusion in this equation. Thisis to be used for the first three samples. For all other samples, alwaysuse the average peak area of the next four bracketed monosaccharidestandards and the previous four bracketed monosaccharide standards formolar ratio calculations.

Calculate the molar ratio of Fucose/Protein:

$R_{Fuc} = \frac{A_{Fuc} \times A_{{Xy}\; 10} \times V_{{Fuc}\; 0} \times C_{{Fuc}\; 0} \times {MW}_{{CTLA}\; 4\text{-}{Ig}}}{A_{{Xy}\; 1} \times A_{{Fuc}\; 0} \times V_{p} \times C_{p} \times {MW}_{Fuc}}$

-   -   Where:    -   R_(Fuc)=molar ratio of Fucose (Fuc) vs. protein    -   A_(Fuc)=peak area (μV·sec) of Fuc in sample    -   A_(Xyl)=peak area (μV·sec) of Xylose (Xyl) in sample    -   A_(Xyl0)=peak area (μV·sec) average of Xyl in monosaccharide        standard    -   A_(Fuc0)=peak area (μV·sec) average of Fuc in monosaccharide        standard    -   V_(Fuc0)=volume of Fuc contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(Fuc0)=concentration of Fuc contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig Reference Material as        per Certificate of Analysis (COA)    -   MW_(Fuc)=Molecular weight of Fucose (164.2 daltons)

Calculate the molar ratio of Galactose/Protein:

$R_{Gal} = \frac{A_{Gal} \times A_{{Xy}\; 10} \times V_{{Gal}\; 0} \times C_{{Gal}\; 0} \times {MW}_{{CTLA}\; 4\text{-}{Ig}}}{A_{{Xy}\; 1} \times A_{{Gal}\; 0} \times V_{P} \times C_{P} \times {MW}_{Gal}}$

-   -   Where:    -   R_(Gal)=molar ratio of Galactose (Gal) vs. protein    -   A_(Gal)=peak area (μV·sec) of Gal in sample    -   A_(Xyl)=peak area (μV·sec) of Xylose (Xyl) in sample    -   A_(Xyl0)=peak area (μV·sec) average of Xyl in monosaccharide        standard A_(Gal0)    -   V_(Gal0)=volume of Gal contained in monosaccharide working        solution used for hydrolysis (in μL)    -   C_(Gal0): =concentration of Gal contained in monosaccharide        working solution used for hydrolysis (in mg/mL)    -   Vp=volume of protein sample used for hydrolysis (in μL)    -   Cp=concentration of protein sample used for hydrolysis (in        mg/mL)    -   MW_(CTLA4-Ig)=Molecular weight of CTLA4-Ig Reference Material as        per Certificate of Analysis (COA)    -   MW_(Gal)=Molecular weight of Gal (180.2 daltons)

Note: When calculating molar ratios of CTLA4-Ig reference material andsamples, use the last System Suitability Standard and the next bracketedSystem Suitability Standard preparation. Average the peak areas forinclusion in this equation. This is to be used for the first sixsamples. For all other samples, always use the average peak area of thetwo bracketed monosaccharide standards for molar ratio calculations.

Exemplary values. The percent RSD for the two, bracketed, neutral SystemSuitability Standard peak area ratios should not exceed 10%. The averagemolar ratios for neutral monosaccharides in the reference material canbe within the ranges specified in Table below. For each component, the %RSD for the four results (duplicate injections of duplicatepreparations) must be </=25%.

Molar Ratio range of CTLA4-Ig Reference Material Monosaccharide RangeMannose 11-18 Fucose 4.2-7.5 Galactose 9.2-18 

Reporting Results. Report the average result as number of Mannosemolecules per CTLA4-Ig molecule, number of Fucose molecules per CTLA4-Igmolecule, and number of Galactose molecules per CTLA4-Ig molecule.Report molar ratio results to two significant figures. For eachcomponent, the % RSD for the four results (duplicate injections ofduplicate preparations) must be </=25%.

Example 65 Tryptic Mapping Quantitation of CTLA4-Ig Oxidation andDeamidation

The purpose of the method is to monitor the lot-to-lot consistency ofCTLA4-Ig using a manual tryptic peptide mapping procedure with specificdetection and quantitation of methionine oxidation and asparaginedeamidation. Peptide mapping involves the proteolysis or otherfragmentation of a protein to create a well-defined set of peptidefragments which are then analyzed, usually by HPLC. The chromatogram orpeptide map is very sensitive to even the smallest change in thechemical structure of the protein and is thus useful for detecting andcharacterizing posttranslational modifications. The CTLA4-Ig proteinsample is denatured in 8 M guanidine-HCl buffer and the cystinedisulfide bridges reduced with dithiothreitol and S-alkylated withiodoacetamide prior to digestion with the proteolytic enzyme, trypsin.The resulting mixture of tryptic peptides is then analyzed by reversedphase high performance liquid chromatography (RP-HPLC) with UV detectionat 215 and 280 nm. Some abbreviations are listed below:

Asn Asparagine Asp Aspartic Acid Asu Aminosuccinimide isoAsp IsoasparticAcid Met(O) Methionine Sulfoxide T26 (281-302) tryptic peptide T26deam1isoAsp294 (281-302) tryptic peptide T26deam2 Asp299 (281-302) trypticpeptide T26deam3 Asp294 (281-302) tryptic peptide T26deam4 Asu294(281-302) tryptic peptide T6 (84-93) tryptic peptide T6oxMet(O)85(84-93) tryptic peptide

Chemicals and Reagents

Mobile Phase A—0.1% TFA in HPLC grade water

-   -   Transfer entire contents of a 1 mL ampule of TFA to 1000 mL of        HPLC grade water and mix thoroughly to prepare 0.1% TFA (Mobile        Phase A). The 0.1% TFA may be stored at room temperature for up        to two months.

Mobile Phase B—0.1% TFA in 80% ACN and 20% HPLC grade water

-   -   Transfer entire contents of a 1 mL ampule of TFA to 800 mL of        acetonitrile and 200 mL HPLC grade water and mix thoroughly to        prepare 0.1% TFA in 80% ACN (Mobile Phase B) which may be stored        at room temperature for up to two months.

Dilution Buffer—100 mM TRIS, 25 mM NaCl, pH 7.6

-   -   Dissolve 14.0 g Trizma Pre-Set Crystal pH 7.6 and 1.46 g NaCl in        1000 mL of HPLC grade water by stirring the solution on a        magnetic stir plate. Filter the solution through 0.2 μm filter        unit. Store solution at 2 to 8° C. for up to two months.

Denaturing Buffer—8 M Guanidine, 50 mM TRIS, pH 8.0

-   -   Dissolve 152.8 g guanidine hydrochloride and 1.4 g Trizma        Pre-Set Crystal pH 8 in 90 mL of HPLC grade water by stirring        the solution on a magnetic stir plate. Adjust the pH to 8.0 with        either HCl or NaOH and bring to the final volume of 200 mL with        HPLC grade water. Filter the solution through 0.2 μm filter.        Store solution at room temperature for up to six months.

Digestion Buffer—50 mM TRIS, 10 mM CaCl₂, pH 7.6

-   -   Dissolve 7.0 g Trizma Pre-Set Crystal pH 7.6 and 1.47 g CaCl₂ in        1000 mL of HPLC grade water by stirring the solution on a        magnetic stir plate. Filter the solution through 0.2 μm filter.        Store solution at 2 to 8° C. for up to two months.

Reducing Agent—200 mM dithiothreitol (DTT)

-   -   To 30.8±0.2 mg of DTT, add 1000 μL of water immediately before        use and vortex until dissolved. Solution expires 24 hours from        the time of preparation.

Alkylating Reagent—400 mM iodoacetamide (IAM)

-   -   To 74.0±0.5 mg iodoacetamide, add 1000 μL of water immediately        before use and vortex until dissolved. Solution expires 24 hours        from the time of preparation.

1.0 M HCl

-   -   Dilute 8.7 mL of concentrated hydrochloric acid to 100 mL with        HPLC grade water. Store solution at room temperature for up to        two months.

Standards and Controls

T6ox peptide standard, 30 μM

-   -   The T6ox tryptic peptide synthetic standard is        Ala-Met(O)-Asp-Thr-Gly-Leu-Tyr-Ile-Cys-Lys • 2TFA, FW 1358.4,        ˜95% purity by weight. Store the solid tightly-sealed at −20° C.        and always warm to room temperature in a dessicator to prevent        the absorption of moisture. Weigh out 1.0±0.1 mg of T6ox, record        the exact weight, and dissolve in 1.50 mL of Digestion Buffer.        Add 40 uL of 200 mM DTT and place at 37° C. for 20 min. Cool to        room temperature, add 48 μL of 400 mM iodoacetamide and alkylate        at room temperature in the dark, for 30 min. Dilute to a final        volume of 24.5 mL with Digestion Buffer to give a 30±3 μM        standard solution. Store 1 mL aliquots of the 30 μM T6ox        standard at −70° C. for up to 24 months.

T26deam1 peptide standard, 30 μM

-   -   The T26deam1 tryptic peptide synthetic standard is        Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-Ser-isoAsp-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys        • 2TFA, FW 2773.7, ˜85% purity by weight. Store the solid        tightly-sealed at −20° C. and always warm to room temperature in        a dessicator to prevent the absorption of moisture. Weigh out        1.0±0.1 mg of T26deam1, record the exact weight, and dissolve in        1 mL of 30% v:v acetonitrile in Digestion Buffer. Dilute to a        final volume of 10.7 mL to give a 30±3 μM standard solution.        Store 1 mL aliquots of the 30 μM T26deam1 standard at −70° C.        for up to 24 months.

Preparation of Standards and Samples

Reduction and Alkylation

The protein concentration range for peptide mapping is approximately 20mg/mL. If the protein concentration is >20 mg/mL, dilute the sample withdilution buffer to a final concentration of approximately 20 mg/mL.Prepare at least 100 μL of diluted solution. In a 1.7 mL centrifuge tubeadd 100 μL of 20 mg/mL (2 mg) CTLA4-Ig solution (sample or referencematerial) to 550 μL of Denaturing Buffer. Add 35 μL of 200 mM ReducingReagent, vortex the tube for 3-5 seconds, then centrifuge forapproximately 3 seconds. Incubate the tubes at 37° C. for 20±2 minutes.Add 38.5 μL of 400 mM Alkylating Reagent to each tube, vortex for 3-5seconds, then centrifuge for approximately 3 seconds. Incubate thesamples at room temperature in the dark for 20±2 minutes. Place theNAP-5 columns in a stand. Use one column per sample. While the samplesare incubating in IAM, equilibrate the NAP-5 columns with 7.5 mL ofDigestion Buffer. Discard the effluent per site procedures. Add 500 μLof the reduced and alkylated mixtures over the NAP-5 columns, allowingthe liquid to drain through column. Discard the effluent per siteprocedures. Add 1.0 mL of Digestion Buffer into the NAP-5 columns andcollect the effluent into 1.7 mL centrifuge tubes and gently mix thecollected effluent.

Digestion. Reconstitute one trypsin vial (20 μg) for each mL of sampleor reference material to be digested, plus one additional trypsin vial,with 86 μL each of trypsin buffer (supplied with the enzyme) to a give0.25 μg/μL. Pool the contents of the trypsin vials together into onevial. Digest each sample with 80 μL of the above pooled trypsin solutionper mL of sample at 37° C. for 120±12 minutes. Upon completion of thedigestion, acidify the samples with 40 μL of 1.0 M HCl per mL of sample,and vortex for 3-5 seconds. Pipette 100 μL each of the digests ofsamples and the reference material into autosampler vials. Prepare anadditional system suitability control containing 95 μL of referencematerial digest mixed with 5 μL of 30 μM T6ox peptide standard and 5 μLof 30 μM T26deam1 peptide standard to give a digest spiked with 5% T6oxand 5% T26deam1. Place all vials in the autosampler at 5±5° C. for HPLCanalysis. Store all the remaining digest samples at −70° C.

Chromatographic Conditions

The table below shows the flow rate and the chromatography gradient.

Time Flow Mobile Mobile (min) (mL/min) Phase A Phase B 0 0.25 100 0 40.25 100 0 10 0.25 92 8 72 0.25 72 28 84 0.25 60 40 92 0.25 0 100 940.40 0 100 95 0.40 100 0 109 0.40 100 0 110 0.25 100 0

Equilibrate column at 55° C. with 100% Mobile Phase A for at least 25minutes prior to first injection. Monitor UV absorbance at 215 nm and280 nm. Tubing from column to detector should have an inner diameter of≤0.01″ in order to minimize diffusion band-broadening. Maintain columntemperature at 55±2° C. Maintain autosampler temperature at 5±5° C.Mobile Phase A is used as the blank injection. Bracket samples (not morethan ten at a time) with reference material injections. The table belowshows the injection sequence for the chromatographic analysis. Note thatall injection volumes are 25 μL except for the control sample consistingof Reference Material spiked 5% T6ox and 5% T26deam1 peptide standardsfor which 28 μL is injected:

Vial # Injection Sample Name Inj. Volume (μL) 1 1 Blank (mobile phase A25 2 1 Reference Material 25 3 1 Sample 1 25 4 1 Sample 2 25 5 1 Sample3 25 6 1 Reference Material spiked with 28 5% T6ox and 5% T26deam1 2 1Reference Material 25

Exemplary Values

Exemplary values. The peptide map profile for the reference materialmust be visually comparable to the chromatogram presented in FIG. 1 withregard to number, relative size and elution order of significant peaksfor the 215 nm and 280 nm traces. The retention time differences forpeaks T4, T25, and T27 in the initial and bracketing reference materialshould not exceed±0.5 minutes. Number of Theoretical Plates (N) must be100,000. If N <100,000, re-equilibrate the column. If the problempersists, replace the column. Resolution (R) must be ≥1.5 for the T2 andT12 peaks. If R <1.5, re-equilibrate the column. If the problempersists, replace the column. The 280 nm chromatogram of the referencestandard spiked with 5% T6ox and T6deam1 must show an increase for theT6ox peak eluting at ˜33.0 min, as shown in FIG. 84. The 215 nmchromatogram of the reference standard spiked with 5% T6ox and T6deam1must show an increase for the T26deam1 peak eluting at ˜66.5 min, asshown in FIG. 86.

Sample Exemplary values. The chromatograms of the first referencematerial injection and the sample must be visually equivalent withregard to number, relative size and elution order of significant peaksfor the 215 nm and 280 nm traces as indicated for the labeled peaks inFIG. 84 with the exception oxidized and/or deamidated peaks for T6ox andT26deam which are reported separately. The retention times for peaks T4,T25, and T27 in the sample must be within±0.5 minutes of the retentiontime for the corresponding peaks of the first reference materialinjection.

CALCULATIONS. NOTE: Use the 215 nm data for these calculations unlessotherwise specified. The retention times of peaks T4, T25, and T27 (FIG.84) in the bracketing reference material runs should not differ morethan 0.5 min (FIG. 84).

Number of Theoretical Plates. Column efficiency, evaluated as the numberof theoretical plates (N), is calculated using the retention time andthe width of peak T27 from the reference material run, (FIG. 84),according to the following equation:

$N = {16\left( \frac{t}{w} \right)^{2}}$

-   -   WHERE:    -   w=the peak width at the baseline measured by extrapolating the        relatively straight sides to the baseline.    -   t=the retention time of the peak T27 measured from time of        injection to time of elution of peak maximum.

Resolution. The resolution (R) between peak T12 and peak T2 (FIG. 84) iscalculated using the following equation:

$R = \frac{2\left( {t_{2} - t_{1}} \right)}{\left( {w_{1} + w_{2}} \right)}$

-   -   WHERE:    -   t₁, t₂=retention times of peak T12 and peak T2, respectively    -   w₁, w₂=tangent-defined peak width at baseline of the peaks with        retention times t₁ and t₂, respectively.

For all samples and standards, calculate Percent Oxidation of Met85 fromthe 280 nm peak area data as follows:

Percent Oxidation=100*A_(T6ox)/(A_(T6ox)+A_(T6))

-   -   WHERE:    -   A_(T6)=peak area for T6, (84-93) in 280 nm trace    -   A_(T6ox)=peak area for T6ox, Met(O)⁸⁵(84-93), in 280 nm trace

For all samples and standards, calculate the Percent Deamidation ofAsn294 for the 215 nm peak area as data shown in FIG. 86:

${PercentDeamidation} = {100^{*}\frac{A_{T\; 26{deam}\; 1}}{A_{T\; 26} + A_{T\; 26{deam}\; 1} + A_{T\; 26{deam}\; 2} + A_{T\; 26{deam}\; 3} + A_{T\; 26{deam}\; 4}}}$

-   -   WHERE:    -   A_(T26)=peak area for T26, (281-302), in 215 nm trace    -   A_(T26deam1)=peak area for T26deam1, isoAsp²⁹⁴(281-302), in 215        nm trace    -   A_(T26deam2)=peak area for T26deam2, Asp²⁹⁹(281-302), in 215 nm        trace    -   A_(T26deam3)=peak area for T26deam3, Asp²⁹⁴(281-302), in 215 nm        trace    -   A_(T26deam4)=peak area for T26deam4, Asu²⁹⁴(281-302), in 215 nm        trace        Theoretically Expected Fragments of CTLA4-Ig Tryptic digest        (refer to FIG. 84)

Fragment Residue Sequence T1  1-14 MHVAQPAVVLASSR T2 15-28GIASFVCEYASPGK T3 29-33 ATEVR T4 34-38 VTVLR T5* 39-83QADSQVTEVCAATYMMGNELTFLDDSICTGTSSG NQVNLTIQGLR T6 84-93 AMDTGLYICK T7* 94-128 VELMYPPPYYLGIGNGTQIYVIDPEPCPDSDQE PK T8** 129-132 SSDK T9**133-158 THTSPPSPAPELLGGSSVFLFPPKPK T10 159-165 DTLMISR T11 166-184TPEVTCVVVDVSHEDPEVK T12 185-198 FNWYVDGVEVHNAK T13 199-202 TKPR T14*203-211 EEQYNSTYR T15 212-227 VVSVLTVLHQDWLNGK T16 228-230 EYK T17231-232 CK T18 233-236 VSNK T19 237-244 ALPAPIEK T20 245-248 TISK T21249-250 AK T22 251-254 GQPR T23 255-265 EPQVYTLPPSR T24 266-270 DELTKT25 271-280 NQVSLTCLVK T26 281-302 GFYPSDIAVEWESNGQPENNYK T27 303-319TTPPVLDSDGSFFLYSK T28 320-324 LTVDK T29 325-326 SR T30 327-349WQQGNVFSCSVMHEALHNHYTQK T31 350-356 SLSLSPG *Contains N-linkedcarbohydrate. **Contains O-linked carbohydrate.

Example 66 Healthy Single Dose Human Study

A single site, randomized, single-dose, study was used to evaluate thepharmacokinetics of CTLA4-Ig (produced by the CD-CHO1) process inhealthy subjects. Thirteen (13) subjects who fulfilled the Inclusion andExclusion Exemplary values were admitted to the clinical study unit andreceived CTLA4-Ig produced by Process CD-CHO1 as a single intravenousinfusion of 10 mg/kg over 30 minutes. Each subject was observed in theclinical study unit for 24 hours following the infusion. Blood sampleswere collected at specified time points after dosing for up to 71 daysfor quantitation of CTLA4-Ig. The subjects were evaluated as topharmacokinetics: Cmax, Tmax, AUC (INF), T-HALF, CLT, and Vss for eachsubject were derived from serum concentration versus time data. CTLA4-Igwas supplied as a 200 mg/vial formulation. Healthy subjects wereadministered a 30-minute IV infusion of 10 mg/kg CTLA4-Ig.Determinations of PK, safety, and immunogenic determinations wereassessed at specified time points through Day 71 after dosing.

Statistical Methods:

Sample Size: The sample size of 13 subjects provided 90% confidence thatthe estimate of the ratio of geometric mean would be within 15% of thetrue value for Cmax, and within 10% of the true value for AUC(INF) forCTLA4-Ig. Statistical Analysis: Subject demographics, physicalexaminations, laboratory data, and vital signs were summarized.Incidence of adverse events was tabulated by body system and severity.For Cmax and AUC(INF) of CTLA4-Ig, 90% confidence intervals for theratios of population geometric means for the Process CD-CHO1 werecalculated from the results of an analysis of variance on log(Cmax) andlog(AUC).

PHARMACOKINETIC RESULTS: The pharmacokinetic results were determinedusing a validated noncompartmental analysis program. Pharmacokineticparameters were obtained from 13 subjects dosed with Process CD-CHO1CTLA4-Ig. The following table lists the pharmacokinetic parameters forCTLA4-Ig in healthy subjects. The Table below shows the summarystatistics for the CTLA4-Ig pharmacokinetic parameters.

Pharmacokinetic Paramter (N = 13) Cmax (μg/mL) Geometric Mean 284.7 (CV%) (23%) AUC (INF) (μg · h/mL) Geometric Mean 44403.0 (CV %) (18%) Tmax(h) Median 0.50 (min, max) (0.50, 2.00) T-HALF (Days) Mean 16.68 (SD)(3.24) CLT (mL/h/kg) Mean 0.23 (SD) (0.04) Vss (L/kg) Mean 0.09 (SD)(0.02)

CTLA4-Ig had mean T-HALF values of approximately 17 days in healthysubjects, consistent with half lives obtained in psoriasis subjects(10-18 days) and rheumatoid arthritis patients (approximately 13 days).The observed mean Vss values of 0.09 to 0.10 L/kg indicated thatCTLA4-Ig was confined primarily to the vascular system and did notdistribute significantly into extravascular spaces.

The following table shows the serum concentrations (ng/ml) per subject.

Serum Assay for CTLA4-Ig

Serum samples were analyzed for CTLA4-Ig by an enzyme-linkedimmunosorbent assay (ELISA) in a total of 25 analytical runs. Allanalytical results met the exemplary values established prior to sampleanalysis indicating that the ELISA method was precise and accurate forthe quantitation of CTLA4-Ig in study samples. A summary of the standardcurve parameters and mean QC data for CTLA4-Ig in serum are presented inTable 48. The between-and within-run variability of the analytical QCsfor CTLA4-Ig was 4.5% and 3.5% CV, respectively. Mean observedconcentrations of the analytical QC samples deviated less than±8.9% fromthe nominal values (Table 48).

TABLE 48 Summary of Quality Control Data for the Assay of CTLA4-Ig inHuman Serum Low Mid High Nominal Conc. (3,000 ng/mL) (12,500 ng/mL)(24,000 ng/mL) Mean Observed Conc. 2.866 13.608 24.526 % Dev. −4.5 8.92.2 Between Run 4.5 2.8 3.0 Precision (% CV) Within Run 2.4 3.5 2.9Precision (% CV) Total Variation 5.1 4.5 4.2 (% CV) N 75 75 75 Number ofRuns 25 25 25

Pharmacokinetics of CTLA4-Ig

The mean and standard deviations for CTLA4-Ig serum concentrations forall subjects by process are presented in the Table directly below. Themean CTLA4-Ig serum concentrations versus time profiles over 71 days arepresented in FIG. 43.

Mean Serum Concentration vs. Time Data for CTLA4-Ig (ng/ml) Day Hr Min NMean SD % CV . . 0 15 0.42 0.87 207.21 . . 15 15 135475.0 28811.82 21.27. . 30 15 273867.9 71406.26 26.07 . 1 0 15 253311.5 43221.58 17.06 . 2 015 254479.4 39611.12 15.57 . 6 0 15 219082.5 44894.29 20.49 . 12 0 15191885.0 45180.00 23.55 1 0 0 15 161732.2 28740.25 17.77 3 0 0 15101411.4 18615.59 18.36 7 0 0 15 59375.96 13598.10 22.90 14 0 0 1533676.97 8148.64 24.20 21 0 0 15 21909.72 5226.77 23.86 28 0 0 1517193.52 5145.61 29.93 42 0 0 15 8828.95 3246.22 36.77 56 0 0 15 5244.512621.36 49.98 70 0 0 15 2970.32 1811.64 60.99

Summary statistics for the pharmacokinetic parameters (Cmax, AUC (INF),CLT, Vss, Tmax, and T-HALF) are presented in Table 50. The resultsindicated that CTLA4-Ig produced from a process of the invention had amean T-HALF value of approximately 17 days (range from 7-25 days). Theobserved Vss values of 0.09 to 0.10 L/kg indicated that CTLA4-Ig wasconfined primarily to the extracellular fluid volume.

TABLE 50 Summary Statistics for Pharmacokinetic Parameters of CTLA4-Igproduced by the process of the invention Cmax AUC(INF) Clearance VssTmax T-HALF (μg/mL) (μg · h/mL) (mL/h/kg) (L/kg) (h) (Days) Geom. MeanGeom. Mean Mean Mean Median Mean Formulation (CV %) (CV %) (SD) (SD)(min, max) (SD) Process 284.71 44403.04 0.23 0.09 0.50 16.68 (n = 13)(23%) (185) (0.04) (0.02) (0.50, 2.00) (3.24)The results indicated that the mean T-HALF for CTLA4-Ig produced byprocess of the invention was approximately 17 days. Both clearance andvolume of distribution values are also presented in Table 50.

Pharmacokinetics of CTLA4-Ig in healthy adult subjects after a single 10mg/kg ntravenous infusion and in RA patients after multiple 10 mg/kgintravenous infusions are set) in Table 47.

TABLE 47 Pharmacokinetic Parameters (Mean, Range) in Healthy Subjectsand RA Patients After 10 mg/kg Intravenous Infusion(s) RA PatientsHealthy Subjects (After 10 mg/kg (After 10 mg/kg Single Multiple PKParameter Dose n = 13) Doses^(a)) n = 14) Peak Concentration 292(175-427) 295 (171-398 (C_(max)) [mcg/mL] Terminal half-life (t_(1/2))16.7 (12-23) 13.1 (8-25) [days] Systemic clearance (CL) 0.23 (0.16-0.30)0.22 (0.13-0.47) [mL/h/kg] Volume of distribution 0.09 (0.06-0.13) 0.07(0.02-0.13) (Vss) [L/kg] ^(a)Multiple intravenous infusions wereadministered at days 1, 15, 30, and monthly thereafter.

Example 67 DNA Sequence of Plasmid pcSDhuCTLA4Ig

Bg1II ~~~~~ 1 GATCTCCCGA TCCCCTATGG TCGACTCTCA GTACAATCTG CTCTGATGCCGCATAGTTAA CTAGAGGGCT AGGGGATACC AGCTGAGAGT CATGTTAGAC GAGACTACGGCGTATCAATT 61 GCCAGTATCT GCTCCCTGCT TGTGTGTTGG AGGTCGCTGA GTAGTGCGCGAGCAAAATTT CGGTCATAGA CGAGGGACGA ACACACAACC TCCAGCGACT CATCACGCGCTCGTTTTAAA 121 AAGCTACAAC AAGGCAAGGC TTGACCGACA ATTGCATGAA GAATCTGCTTAGGGTTAGGC TTCGATGTTG TTCCGTTCCG AACTGGCTGT TAACGTACTT CTTAGACGAATCCCAATCCG                  ?-----------------CMVPromoter------------------- 181 GTTTTGCGCT GCTTCGCGAT GTACGGGCCAGATATACGCG TTGACATTGA TTATTGACTA CAAAACGCGA CGAAGCGCTA CATGCCCGGTCTATATGCGC AACTGTAACT AATAACTGAT----------------------------------------------------------------- 241GTTATTAATA GTAATCAATT ACGGGGTCAT TAGTTCATAG CCCATATATG GAGTTCCGCGCAATAATTAT CATTAGTTAA TGCCCCAGTA ATCAAGTATC GGGTATATAC CTCAAGGCGC----------------------------------------------------------------- 301TTACATAACT TACGGTAAAT GGCCCGCCTG GCTGACCGCC CAACGACCCC CGCCCATTGAAATGTATTGA ATGCCATTTA CCGGGCGGAC CGACTGGCGG GTTGCTGGGG GCGGGTAACT----------------------------------------------------------------- 361CGTCAATAAT GACGTATGTT CCCATAGTAA CGCCAATAGG GACTTTCCAT TGACGTCAATGCAGTTATTA CTGCATACAA GGGTATCATT GCGGTTATCC CTGAAAGGTA ACTGCAGTTA----------------------------------------------------------------- 421GGGTGGACTA TTTACGGTAA ACTGCCCACT TGGCAGTACA TCAAGTGTAT CATATGCCAACCCACCTGAT AAATGCCATT TGACGGGTGA ACCGTCATGT AGTTCACATA GTATACGGTT----------------------------------------------------------------- 481GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT GCCCAGTACACATGCGGGGG ATAACTGCAG TTACTGCCAT TTACCGGGCG GACCGTAATA CGGGTCATGT                                                           NcoI                                                            ~~~----------------------------------------------------------------- 541TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC GCTATTACCAACTGGAATAC CCTGAAAGGA TGAACCGTCA TGTAGATGCA TAATCAGTAG CGATAATGGT NcoI~~~ ---------------------------CMV Promoter---------------------------601 TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC TCACGGGGATACCACTACGC CAAAACCGTC ATGTAGTTAC CCGCACCTAT CGCCAAACTG AGTGCCCCTA----------------------------------------------------------------- 661TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA AATCAACGGGAAGGTTCAGA GGTGGGGTAA CTGCAGTTAC CCTCAAACAA AACCGTGGTT TTAGTTGCCC----------------------------------------------------------------- 721ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT AGGCGTGTACTGAAAGGTTT TACAGCATTG TTGAGGCGGG GTAACTGCGT TTACCCGCCA TCCGCACATG----------------------------------------------------------------- 781GGTGGGAGGT CTATATAAGC ATAGCTCTCT GGCTAACTAG AGAACCCACT GCTTACTGGCCCACCCTCCA GATATATTCG TCTCGAGAGA CCGATTGATC TCTTGGGTGA CGAATGACCG                                       HindIII             BamHI----------->                           ~~~~~~~             ~~~~~~ 841TTATCGAAAT TAATACGACT CACTATAGGG AGACCCAAGC TTGGTACCGA GCTCGGATCCAATAGCTTTA ATTATGCTGA GTGATATCCC TCTGGGTTCG AACCATGGCT CGAGCCTAGG                                  PstI                            EcoRI~~~~~~             ?-huCTLA-4 Ig-                           ~~~~~~~                   M   G  V  L 901ACTAGTAACG GCCGCCAGTG TGCTGGAATT CTGCAGATAG CTTCACCAAT GGGTGTACTGTGATCATTGC CGGCGGTCAC ACGACCTTAA GACGTCTATC GAAGTGGTTA CCCACATGAC----------------------------------------------------------------- L  T  Q  R   T  L  L   S  L  V   L  A  L  L   F  P  S   M  A  S 961CTCACACAGA GGACGCTGCT CAGTCTGGTC CTTGCACTCC TGTTTCCAAG CATGGCGAGCGAGTGTGTCT CCTGCGACGA GTCAGACCAG GAACGTGAGG ACAAAGGTTC GTACCGCTCG----------------------------------------------------------------- M  A  M  H   V  A  Q   P  A  V   V  L  A  S   S  R  G   I  A  S 1021ATGGCAATGC ACGTGGCCCA GCCTGCTGTG GTACTGGCCA GCAGCCGAGG CATCGCCAGCTACCGTTACG TGCACCGGGT CGGACGACAC CATGACCGGT CGTCGGCTCC GTAGCGGTCG----------------------------------------------------------------- F  V  C  E   Y  A  S   P  G  K   A  T  E  V   R  V  T   V  L  R 1081TTTGTGTGTG AGTATGCATC TCCAGGCAAA GCCACTGAGG TCCGGGTGAC AGTGCTTCGGAAACACACAC TCATACGTAG AGGTCCGTTT CGGTGACTCC AGGCCCACTG TCACGAAGCC----------------------------huCTLA-4-Ig--------------------------- Q  A  D  S   Q  V  T   E  V  C   A  A  T  Y   M  M  G   N  E  L 1141CAGGCTGACA GCCAGGTGAC TGAAGTCTGT GCGGCAACCT ACATGATGGG GAATGAGTTGGTCCGACTGT CGGTCCACTG ACTTCAGACA CGCCGTTGGA TGTACTACCC CTTACTCAAC----------------------------------------------------------------- T  F  L  D   D  S  I   C  T  G   T  S  S  G   N  Q  V   N  L  T 1201ACCTTCCTAG ATGATTCCAT CTGCACGGGC ACCTCCAGTG GAAATCAAGT GAACCTCACTTGGAAGGATC TACTAAGGTA GACGTGCCCG TGGAGGTCAC CTTTAGTTCA CTTGGAGTGA                  NcoI                  ~~~~~~~----------------------------------------------------------------- I  Q  G  L   R  A  M   D  T  G   L  Y  I  C   K  V  E   L  M  Y 1261ATCCAAGGAC TGAGGGCCAT GGACACGGGA CTCTACATCT GCAAGGTGAA GCTCATGTACTAGGTTCCTG ACTCCCGGTA CCTGTGCCCT GAGATGTAGA CGTTCCACCT CGAGTACATG----------------------------------------------------------------- P  P  P  Y   Y  L  G   I  G  N   G  T  Q  I   Y  V  I   D  P  E 1321CCACCGCCAT ACTACCTGGG CATAGGCAAC GGAACCCAGA TTTATGTAAT TGATCCAGAAGGTGGCGGTA TGATGGACCC GTATCCGTTG CCTTGGGTCT AAATACATTA ACTAGGTCTT----------------------------------------------------------------- P  C  P  D   S  D  Q   E  P  K   S  S  D  K   T  H  T   S  P  P 1381CCGTGCCCAG ATTCTGATCA GGAGCCCAAA TCTTCTGACA AAACTCACAC ATCCCCACCGGGCACGGGTC TAAGACTAGT CCTCGGGTTT AGAAGACTGT TTTGAGTGTG TAGGGGTGGC----------------------------------------------------------------- S  P  A  P   E  L  L   G  G  S   S  V  F  L   F  P  P   K  P  K 1441TCCCCAGCAC CTGAACTCCT GGGGGGATCG TCAGTCTTCC TCTTCCCCCC AAAACCCAAGAGGGGTCGTG GACTTGAGGA CCCCCCTAGC AGTCAGAAGG AGAAGGGGGG TTTTGGGTTC----------------------------------------------------------------- D  T  L  M   I  S  R   T  P  E   V  T  C  V   V  V  D   V  S  H 1501GACACCCTCA TGATCTCCCG GACCCCTGAG GTCACATGCG TGGTGGTGGA CGTGAGCCACCTGTGGGAGT ACTAGAGGGC CTGGGGACTC CAGTGTACGC ACCACCACCT GCACTCGGTG----------------------------------------------------------------- E  D  P  E   V  K  F   N  W  Y   V  D  G  V   E  V  H   N  A  K 1561GAAGACCCTG AGGTCAAGTT CAACTGGTAC GTGGACGGCG TGGAGGTGCA TAATGCCAAGCTTCTGGGAC TCCAGTTCAA GTTGACCATG CACCTGCCGC ACCTCCACGT ATTACGGTTC---------------------------huCTLA-4 Ig---------------------------- T  K  P  R   E  E  Q   Y  N  S   T  Y  R  V   V  S  V   L  T  V 1621ACAAAGCCGC GGGAGGAGCA GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTCTGTTTCGGCG CCCTCCTCGT CATGTTGTCG TGCATGGCAC ACCAGTCGCA GGAGTGGCAG----------------------------------------------------------------- L  H  Q  D   W  L  N   G  K  E   Y  K  C  K   V  S  N   K  A  L 1681CTGCACCAGG ACTGGCTGAA TGGCAAGGAG TACAAGTGCA AGGTCTCCAA CAAAGCCCTCGACGTGGTCC TGACCGACTT ACCGTTCCTC ATGTTCACGT TCCAGAGGTT GTTTCGGGAG----------------------------------------------------------------- P  A  P  I   E  K  T   I  S  K   A  K  G  Q   P  R  E   P  Q  V 1741CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA ACCACAGGTGGGTCGGGGGT AGCTCTTTTG GTAGAGGTTT CGGTTTCCCG TCGGGGCTCT TGGTGTCCAC                  SmaI                  ~~~~~~~----------------------------------------------------------------- Y  T  L  P   P  S  R   D  E  L   T  K  N  Q   V  S  L   T  C  L 1801TACACCCTGC CCCCATCCCG GGATGAGCTG ACCAAGAACC AGGTCAGCCT GACCTGCCTGATGTGGGACG GGGGTAGGGC CCTACTCGAC TGGTTCTTGG TCCAGTCGGA CTGGACGGAC----------------------------------------------------------------- V  K  G  F   Y  P  S   D  I  A   V  E  W  E   S  N  G   Q  P  E 1861GTCAAAGGCT TCTATCCCAG CGACATCGCC GTGGAGTGGG AGAGCAATGG GCAGCCGGAGCAGTTTCCGA AGATAGGGTC GCTGTAGCGG CACCTCACCC TCTCGTTACC CGTCGGCCTC----------------------------------------------------------------- N  N  Y  K   T  T  P   P  V  L   D  S  D  G   S  F  F   L  Y  S 1921AACAACTACA AGACCACGCC TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGCTTGTTGATGT TCTGGTGCGG AGGGCACGAC CTGAGGCTGC CGAGGAAGAA GGAGATGTCG----------------------------------------------------------------- K  L  T  V   D  K  S   R  W  Q   Q  G  N  V   F  S  C   S  V  M 1981AAGCTCACCG TGGACAAGAG CAGGTGGCAG CAGGGGAACG TCTTCTCATG CTCCGTGATGTTCGAGTGGC ACCTGTTCTC GTCCACCGTC GTCCCCTTGC AGAAGAGTAC GAGGCACTAC----------------------------------------------------------------- H  E  A  L   H  N  H   Y  T  Q   K  S  L  S   L  S  P  G  K  * 2041CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCTCC GGGTAAATGAGTACTCCGAG ACGTGTTGGT GATGTGCGTC TTCTCGGAGA GGGACAGAGG CCCATTTACT                             SmaI                            XbaI                            ~~~~~~~                          ~~~~ 2101GTGCGACGGC CGGCAAGCCC CCGCTCCCCG GGCTCTCGCG GTCGCACGAG GATGCTTCTACACGCTGCCG GCCGTTCGGG GGCGAGGGGC CCGAGAGCGC CAGCGTGCTC CTACGAAGAT XbaI~~                                ?----BGH polyadenylation signal------2161 GAGGGCCCTA TTCTATAGTG TCACCTAAAT GCTAGAGCTC GCTGATCAGC CTCGACTGTGCTCCCGGGAT AAGATATCAC AGTGGATTTA CGATCTCGAG CGACTAGTCG GAGCTGACAC----------------------------------------------------------------- 2221CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAAGGAAGATCAA CGGTCGGTAG ACAACAAACG GGGAGGGGGC ACGGAAGGAA CTGGGACCTT----------------------------------------------------------------- 2281GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGTCCACGGTGAG GGTGACAGGA AAGGATTATT TTACTCCTTT AACGTAGCGT AACAGACTCA----------------------------------------------------------------- 2341AGGTGTCATT CTATTCTGGG GGGTGGGGTG GGGCAGGACA GCAAGGGGGA GGATTGGGAATCCACAGTAA GATAAGACCC CCCACCCCAC CCCGTCCTGT CGTTCCCCCT CCTAACCCTT------------------------> 2401 GACAATAGCA GGCATGCTGG GGATGCGGTGGGCTCTATGG CTTCTGAGGC GGAAAGAACC CTGTTATCGT CCGTACGACC CCTACGCCACCCGAGATACC GAAGACTCGG CCTTTCTTGG 2461 AGCTGGGGCT CTAGGGGGTA TCCCCACGCGCCCTGTAGCG GCGCATTAAG CGCGGCGGGT TCGACCCCGA GATCCCCCAT AGGGGTGCGCGGGACATCGC CGCGTAATTC GCGCCGCCCA 2521 GTGGTGGTTA CGCGCAGCGT GACCGCTACACTTGCCAGCG CCCTAGCGCC CGCTCCTTTC CACCACCAAT GCGCGTCGCA CTGGCGATGTGAACGGTCGC GGGATCGCGG GCGAGGAAAG 2581 GCTTTCTTCC CTTCCTTTCT CGCCACGTTCGCCCTGTGGA ATGTGTGTCA GTTAGGGTGT CGAAAGAAGG GAAGGAAAGA GCGGTGCAAGCGGGACACCT TACACACAGT CAATCCCACA 2641 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGAAGTATGCAAA GCATGCATCT CAATTAGTCA CCTTTCAGGG GTCCGAGGGG TCGTCCGTCTTCATACGTTT CGTACGTAGA GTTAATCAGT 2701 GCAACCAGGT GTGGAAAGTC CCCAGGCTCCCCAGCAGGCA GAAGTATGCA AAGCATGCAT CGTTGGTCCA CACCTTTCAG GGGTCCGAGGGGTCGTCCGT CTTCATACGT TTCGTACGTA 2761 CTCAATTAGT CAGCAACCAT AGTCCCGCCCCTAACTCCGC CCATCCCGCC CCTAACTCCG GAGTTAATCA GTCGTTGGTA TCAGGGCGGGGATTGAGGCG GGTAGGGCGG GGATTGAGGC                           NcoI                         ~~~~~~      ?------SV40 Promoter---------- 2821CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCCGGGTCAAGGC GGGTAAGAGG CGGGGTACCG ACTGATTAAA AAAAATAAAT ACGTCTCCGG--------------------------------------------------------------> 2881GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTACTCCGGCGGA GCCGGAGACT CGATAAGGTC TTCATCACTC CTCCGAAAAA ACCTCCGGAT            HindIII              ~~~~~~ 2941 GGCTTTTGCA AAAAGCTTGGACAGCTGAGG GCTGCGATTT CGCGCCAAAC TTGACGGCAA CCGAAAACGT TTTTCGAACCTGTCGACTCC CGACGCTAAA GCGCGGTTTG AACTGCCGTT                                               -------dhfr-------- 3001TCCTAGCGTG AAGGCTGGTA GGATTTTATC CCCGCTGCCA TCATGGTTCG ACCATTGAACAGGATCGCAC TTCCGACCAT CCTAAAATAG GGGCGACGGT AGTACCAAGC TGGTAACTTG----------------------------------------------------------------- 3061TGCATCGTCG CCGTGTCCCA AGATATGGGG ATTGGCAAGA ACGGAGACCT ACCCTGGCCTACGTAGCAGC GGCACAGGGT TCTATACCCC TAACCGTTCT TGCCTCTGGA TGGGACCGGA----------------------------------------------------------------- 3121CCGCTCAGGA ACGAGTTCAA GTACTTCCAA AGAATGACCA CAACCTCTTC AGTGGAAGGTGGCGAGTCCT TGCTCAAGTT CATGAAGGTT TCTTACTGGT GTTGGAGAAG TCACCTTCCA----------------------------------------------------------------- 3181AAACAGAATC TGGTGATTAT GGGTAGGAAA ACCTGGTTCT CCATTCCTGA GAAGAATCGATTTGTCTTAG ACCACTAATA CCCATCCTTT TGGACCAAGA GGTAAGGACT CTTCTTAGCT----------------------------------------------------------------- 3241CCTTTAAAGG ACAGAATTAA TATAGTTCTC AGTAGAGAAC TCAAAGAACC ACCACGAGGAGGAAATTTCC TGTCTTAATT ATATCAAGAG TCATCTCTTG AGTTTCTTGG TGGTGCTCCT----------------------------- dhfr -------------------------------- 3301GCTCATTTTC TTGCCAAAAG TTTGGATGAT GCCTTAAGAC TTATTGAACA ACCGGAATTGCGAGTAAAAG AACGGTTTTC AAACCTACTA CGGAATTCTG AATAACTTGT TGGCCTTAAC----------------------------------------------------------------- 3361GCAAGTAAAG TAGACATGGT TTGGATAGTC GGAGGCAGTT CTGTTTACCA GGAAGCCATGCGTTCATTTC ATCTGTACCA AACCTATCAG CCTCCGTCAA GACAAATGGT CCTTCGGTAC----------------------------------------------------------------- 3421AATCAACCAG GCCACCTCAG ACTCTTTGTG ACAAGGATCA TGCAGGAATT TGAAAGTGACTTAGTTGGTC CGGTGGAGTC TGAGAAACAC TGTTCCTAGT ACGTCCTTAA ACTTTCACTG----------------------------------------------------------------- 3481ACGTTTTTCC CAGAAATTGA TTTGGGGAAA TATAAACTTC TCCCAGAATA CCCAGGCGTCTGCAAAAAGG GTCTTTAACT AAACCCCTTT ATATTTGAAG AGGGTCTTAT GGGTCCGCAG----------------------------------------------------------------- 3541CTCTCTGAGG TCCAGGAGGA AAAAGGCATC AAGTATAAGT TTGAAGTCTA CGAGAAGAAAGAGAGACTCC AGGTCCTCCT TTTTCCGTAG TTCATATTCA AACTTCAGAT GCTCTTCTTT -->3601 GACTAACAGG AAGATGCTTT CAAGTTCTCT GCTCCCCTCC TAAAGCTATG CATTTTTATACTGATTGTCC TTCTACGAAA GTTCAAGAGA CGAGGGGAGG ATTTCGATAC GTAAAAATAT    NcoI                   Bg1II    ~~~~~~                  ~~~~~~~ 3661AGACCATGGG ACTTTTGCTG GCTTTAGATC TTTGTGAAGG AACCTTACTT CTGTGGTGTGTCTGGTACCC TGAAAACGAC CGAAATCTAG AAACACTTCC TTGGAATGAA GACACCACAC 3721ACATAATTGG ACAAACTACC TACAGAGATT TAAAGCTCTA AGGTAAATAT AAAATTTTTATGTATTAACC TGTTTGATGG ATGTCTCTAA ATTTCGAGAT TCCATTTATA TTTTAAAAAT 3781AGTGTATAAT GTGTTAAACT ACTGATTCTA ATTGTTTGTG TATTTTAGAT TCCAACCTATTCACATATTA CACAATTTGA TGACTAAGAT TAACAAACAC ATAAAATCTA AGGTTGGATA 3841GGAACTGATG AATGGGAGCA GTGGTGGAAT GCCTTTAATG AGGAAAACCT GTTTTGCTCACCTTGACTAC TTACCCTCGT CACCACCTTA CGGAAATTAC TCCTTTTGGA CAAAACGAGT 3901GAAGAAATGC CATCTAGTGA TGATGAGGCT ACTGCTGACT CTCAACATTC TACTCCTCCACTTCTTTACG GTAGATCACT ACTACTCCGA TGACGACTGA GAGTTGTAAG ATGAGGAGGT 3961AAAAAGAAGA GAAAGGTAGA AGACCCCAAG GACTTTCCTT CAGAATTGCT AAGTTTTTTGTTTTTCTTCT CTTTCCATCT TCTGGGGTTC CTGAAAGGAA GTCTTAACGA TTCAAAAAAC 4021AGTCATGCTG TGTTTAGTAA TAGAACTCTT GCTTGCTTTG CTATTTACAC CACAAAGGAATCAGTACGAC ACAAATCATT ATCTTGAGAA CGAACGAAAC GATAAATGTG GTGTTTCCTT 4081AAAGCTGCAC TGCTATACAA GAAAATTATG GAAAAATATT CTGTAACCTT TATAAGTAGGTTTCGACGTG ACGATATGTT CTTTTAATAC CTTTTTATAA GACATTGGAA ATATTCATCC 4141CATAACAGTT ATAATCATAA CATACTGTTT TTTCTTACTC CACACAGGCA TAGAGTGTCTGTATTGTCAA TATTAGTATT GTATGACAAA AAAGAATGAG GTGTGTCCGT ATCTCACAGA 4201GCTATTAATA ACTATGCTCA AAAATTGTGT ACCTTTAGCT TTTTAATTTG TAAAGGGGTTCGATAATTAT TGATACGAGT TTTTAACACA TGGAAATCGA AAAATTAAAC ATTTCCCCAA 4261AATAAGGAAT ATTTGATGTA TAGTGCCTTG ACTAGAGATC ATAATCAGCC ATACCACATTTTATTCCTTA TAAACTACAT ATCACGGAAC TGATCTCTAG TATTAGTCGG TATGGTGTAA 4321TGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAAACATCTCCAA AATGAACGAA ATTTTTTGGA GGGTGTGGAG GGGGACTTGG ACTTTGTATT 4381ATTGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAGTTACTTACGT TAACAACAAC AATTGAACAA ATAACGTCGA ATATTACCAA TGTTTATTTC 4441CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTTGTTATCGTAG TGTTTAAAGT GTTTATTTCG TAAAAAAAGT GACGTAAGAT CAACACCAAA 4501GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCGGC TGGATGATCC TCCAGCGCGGCAGGTTTGAG TAGTTACATA GAATAGTACA GACCTAGCCG ACCTACTAGG AGGTCGCGCC 4561GGATCTCATG CTGGAGTTCT TCGCCCACCC CAACTTGTTT ATTGCAGCTT ATAATGGTTACCTAGAGTAC GACCTCAAGA AGCGGGTGGG GTTGAACAAA TAACGTCGAA TATTACCAAT 4621CAAATAAAGC AATAGCATCA CAAATTTCAC AAATAAAGCA TTTTTTTCAC TGCATTCTAGGTTTATTTCG TTATCGTAGT GTTTAAAGTG TTTATTTCGT AAAAAAAGTG ACGTAAGATC 4681TTGTGGTTTG TCCAAACTCA TCAATGTATC TTATCATGTC TGTATACCGT CGACCTCTAGAACACCAAAC AGGTTTGAGT AGTTACATAG AATAGTACAG ACATATGGCA GCTGGAGATC 4741CTAGAGCTTG GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCACGATCTCGAAC CGCATTAGTA CCAGTATCGA CAAAGGACAC ACTTTAACAA TAGGCGAGTG 4801AATTCCACAC AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGTTTAAGGTGTG TTGTATGCTC GGCCTTCGTA TTTCACATTT CGGACCCCAC GGATTACTCA 4861GAGCTAACTC ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTCCTCGATTGAG TGTAATTAAC GCAACGCGAG TGACGGGCGA AAGGTCAGCC CTTTGGACAG 4921GTGCCAGCTG CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCGCACGGTCGAC GTAATTACTT AGCCGGTTGC GCGCCCCTCT CCGCCAAACG CATAACCCGC 4981CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGTGAGAAGGCGA AGGAGCGAGT GACTGAGCGA CGCGAGCCAG CAAGCCGACG CCGCTCGCCA 5041ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAATAGTCGAGTG AGTTTCCGCC ATTATGCCAA TAGGTGTCTT AGTCCCCTAT TGCGTCCTTT    ?------------------------ColE1 ori----------------------------- 5101GAACATGTGA GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGCCTTGTACACT CGTTTTCCGG TCGTTTTCCG GTCCTTGGCA TTTTTCCGGC GCAACGACCG----------------------------------------------------------------- 5161GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAGCAAAAAGGTA TCCGAGGCGG GGGGACTGCT CGTAGTGTTT TTAGCTGCGA GTTCAGTCTC----------------------------------------------------------------- 5221GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGTCACCGCTTTG GGCTGTCCTG ATATTTCTAT GGTCCGCAAA GGGGGACCTT CGAGGGAGCA---------------------------ColE1 ori------------------------------- 5281GCGCTCTCCT GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGGCGCGAGAGGA CAAGGCTGGG ACGGCGAATG GCCTATGGAC AGGCGGAAAG AGGGAAGCCC----------------------------------------------------------------- 5341AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCGTTCGCACCGC GAAAGAGTTA CGAGTGCGAC ATCCATAGAG TCAAGCCACA TCCAGCAAGC                 ApaLI                  ~~~~~~~----------------------------------------------------------------- 5401CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGGGAGGTTCGAC CCGACACACG TGCTTGGGGG GCAAGTCGGG CTGGCGACGC GGAATAGGCC----------------------------------------------------------------- 5461TAACTATCGT CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCACATTGATAGCA GAACTCAGGT TGGGCCATTC TGTGCTGAAT AGCGGTGACC GTCGTCGGTG----------------------------------------------------------------- 5521TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTGACCATTGTCC TAATCGTCTC GCTCCATACA TCCGCCACGA TGTCTCAAGA ACTTCACCAC----------------------------------------------------------------- 5581GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGTCGGATTGATG CCGATGTGAT CTTCCTGTCA TAAACCATAG ACGCGAGACG ACTTCGGTCA----------------------------------------------------------------- 5641TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGGATGGAAGCCT TTTTCTCAAC CATCGAGAAC TAGGCCGTTT GTTTGGTGGC GACCATCGCC----------------------------------------------------------------- 5701TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCCACCAAAAAAA CAAACGTTCG TCGTCTAATG CGCGTCTTTT TTTCCTAGAG TTCTTCTAGG------------------? 5761 TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAAAACTCACGTT AAGGGATTTT AAACTAGAAA AGATGCCCCA GACTGCGAGT CACCTTGCTTTTGAGTGCAA TTCCCTAAAA 5821 GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTTTTAAATTAAA AATGAAGTTT CCAGTACTCT AATAGTTTTT CCTAGAAGTG GATCTAGGAAAATTTAATTT TTACTTCAAA                                                 <-----ampR------ 5881TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAGATTTAGTTAG ATTTCATATA TACTCATTTG AACCAGACTG TCAATGGTTA CGAATTAGTC----------------------------------------------------------------- 5941TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGTACTCCGTGGA TAGAGTCGCT AGACAGATAA AGCAAGTAGG TATCAACGGA CTGAGGGGCA----------------------------------------------------------------- 6001CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACCGCACATCTAT TGATGCTATG CCCTCCCGAA TGGTAGACCG GGGTCACGAC GTTACTATGG----------------------------------------------------------------- 6061GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGCCGCTCTGGGT GCGAGTGGCC GAGGTCTAAA TAGTCGTTAT TTGGTCGGTC GGCCTTCCCG----------------------------------------------------------------- 6121CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCGGCTCGCGTCT TCACCAGGAC GTTGAAATAG GCGGAGGTAG GTCAGATAAT TAACAACGGC----------------------------------------------------------------- 6181GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTACCCTTCGATCT CATTCATCAA GCGGTCAATT ATCAAACGCG TTGCAACAAC GGTAACGATG----------------------------------------------------------------- 6241AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACGTCCGTAGCAC CACAGTGCGA GCAGCAAACC ATACCGAAGT AAGTCGAGGC CAAGGGTTGC----------------------------------------------------------------- 6301ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCCTAGTTCCGCT CAATGTACTA GGGGGTACAA CACGTTTTTT CGCCAATCGA GGAAGCCAGG   PvuI   ~~~~~~------------------------------ampR------------------------------- 6361TCCGATCGTT GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACTAGGCTAGCAA CAGTCTTCAT TCAACCGGCG TCACAATAGT GAGTACCAAT ACCGTCGTGA----------------------------------------------------------------- 6421GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTCCGTATTAAGA GAATGACAGT ACGGTAGGCA TTCTACGAAA AGACACTGAC CACTCATGAG----------------------------------------------------------------- 6481AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAATTTGGTTCAGT AAGACTCTTA TCACATACGC CGCTGGCTCA ACGAGAACGG GCCGCAGTTA----------------------------------------------------------------- 6541ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTCTGCCCTATTA TGGCGCGGTG TATCGTCTTG AAATTTTCAC GAGTAGTAAC CTTTTGCAAG----------------------------------------------------------------- 6601TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCACAAGCCCCGCT TTTGAGAGTT CCTAGAATGG CGACAACTCT AGGTCAAGCT ACATTGGGTG  ApaLI  ~~~~~~----------------------------------------------------------------- 6661TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAAAGCACGTGGG TTGACTAGAA GTCGTAGAAA ATGAAAGTGG TCGCAAAGAC CCACTCGTTT----------------------------------------------------------------- 6721AACAGGAAGG CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACTTTGTCCTTCC GTTTTACGGC GTTTTTTCCC TTATTCCCGC TGTGCCTTTA CAACTTATGA --?6781 CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGGGTATGAGAAG GAAAAAGTTA TAATAACTTC GTAAATAGTC CCAATAACAG AGTACTCGCC 6841ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCGTATGTATAAA CTTACATAAA TCTTTTTATT TGTTTATCCC CAAGGCGCGT GTAAAGGGGC                                Bg1II                                  ~6901 AAAAGTGCCA CCTGACGTCG ACGGATCGGG A TTTTCACGGT GGACTGCAGC TGCCTAGCCCT

Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 21,28, 29, 30, 31, 32, 33, 34, 42, 44, 45, 46, 47, 48, 49, 50, 51, 58, 59,60, 61, 62, 63, 64, 65, 66, 67 above pertain in particular to theCTLA4-Ig protein having SEQ ID NO: 1, 2, 5, 6, 7, 8, 9, 10 or 18, andExamples 19, 20, 22, 23, 24, 25, 26, 27, 35, 36, 37, 38, 39, 40, 41, 52,53, 54, 55, 56, 57 above pertain in particular to the CTLA4-Ig proteinhaving SEQ ID NO: 4, 11, 12, 13, 14, 15 or 16. As described in thisspecification, the methods relating to, and uses of, these proteins inthe Examples are illustrative for methods relating to, and uses of,other CTLA4-Ig proteins of the invention.

Exemplary and non-limiting compositions comprising CTLA4-Ig molecules ofthe invention include such compositions wherein:

(1) the CTLA4-Ig molecules comprise any one or more of SEQ ID NO: 2, 5,6, 7, 8, 9, 10 or 18, and the CTLA4-Ig molecules are less than or equalto about 5.0 area percent CTLA4-Ig high molecular weight species (orCTLA4-Ig tetramers) as determined by size exclusion chromatography andspectrophotometric detection (a method of measurement for which is setout in Example 10). More particularly, the invention provides suchcompositions further having one or more of the followingcharacteristics:

not exceeding a maximum amount of bacterial endotoxin of 0.35 EU/mgCTLA4-Ig molecules or 76.8 EU/mL (which can be an absence of bacterialendotoxin); a method for measuring this characteristic is set out inExample 48;

not exceeding a maximum bioburden of 1 CFU/10 mL or 1 CFU/mL (which canbe an absence of bioburden); a method for measuring this characteristicis set out in Example 49;

providing (as CTLA4-Ig molecules or as said composition) (a) about 10 to22 bands with a pI range of about 4.3 to about 5.6, cumulative bandsintensity of 90%-110% at a pI range of about 4.3 to about 5.3, and about3 major bands at a pI range of about 4.5 to about 5.2, or (b) dominantCTLA4-Ig isoforms having a pI that is less than or equal to 5.1, and atleast 90% of the CTLA4-Ig molecules exhibit a pI less than or equal toabout 5.3; a method for measuring this characteristic is set out inExample 50;

less than or equal to 3.5 area percent of the CTLA4-Ig molecules areoxidized species thereof, and less than or equal to 2.5 area percent ofthe CTLA4-Ig molecules are deamidated species thereof; methods formeasuring these characteristics are set out in Example 47;

the CTLA4-Ig molecules are greater than or equal to 95.0 area percentCTLA4-Ig dimers as determined by size exclusion chromatography andspectrophotometric detection; a method for measuring this characteristicis set out in Example 10;

the CTLA4-Ig molecules are less than 5.0 area percent CTLA4-Ig highmolecular weight species (or CTLA4-Ig tetramers) as determined by sizeexclusion chromatography and spectrophotometric detection; a method formeasuring this characteristic is set out in Example 10;

the CTLA4-Ig molecules are less than or equal to 0.5 area percent lowmolecular weight species (or CTLA4-Ig monomers) as determined by sizeexclusion chromatography and spectrophotometric detection, or less than0.5 area percent low molecular weight species (or CTLA4-Ig monomers) asdetermined by size exclusion chromatography and spectrophotometricdetection; a method for measuring this characteristic is set out inExample 10;

not exceeding a maximum amount of DNA of 2.5 picogram/mg CTLA4-Igmolecules or 2.5 picogram/mg CTLA4-Ig dimer (which can be an absence ofDNA); a method for measuring this characteristic is set out in Example58;

not exceeding a maximum amount of MCP-1 of 3.0 ng/mg total CTLA4-Igmolecules, 5 ppm, or 5 ng/mg CTLA4-Ig dimer (which can be an absence ofMCP-1); a method for measuring this characteristic is set out in Example59;

not exceeding a maximum amount of host cell protein (also known ascellular protein) of 25 ng/mg CTLA4-Ig molecules or 50 ng/mg CTLA4-Igdimer (which can be an absence of host cell protein or cellularprotein); a method for measuring this characteristic is set out inExample 60;

not exceeding a maximum amount of Triton X-100 of 1.0 ng/mg CTLA4-Igmolecules (which can be an absence of Triton-X); a method for measuringthis characteristic is set out in Example 61;

not exceeding a maximum amount of Protein A of 5.0 ng/mg CTLA4-Igmolecules (which can be an absence of Protein A); a method for measuringthis characteristic is set out in Example 62;

the CTLA4-Ig molecules have an average molar ratio of GlcNAc to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 15 to about 35; a method for measuring this characteristic isset out in Example 63;

the CTLA4-Ig molecules have an average molar ratio of GalNAc to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 1.7 to about 3.6; a method for measuring this characteristicis set out in Example 63;

the CTLA4-Ig molecules have an average molar ratio of galactose toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of from about 8.0 to about 17; a method for measuring thischaracteristic is set out in Example 64;

the CTLA4-Ig molecules have an average molar ratio of fucose to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 3.5 to about 8.3; a method for measuring this characteristicis set out in Example 64;

the CTLA4-Ig molecules have an average molar ratio of mannose toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of from about 7.7 to about 22; a method for measuring thischaracteristic is set out in Example 64;

the CTLA4-Ig molecules have an average molar ratio of sialic acid toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of greater than or equal to 8.0, such as from about 8.0 toabout 12.0; a method for measuring this characteristic is set out inExample 16;

the CTLA4-Ig molecules have an average molar ratio of NANA to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, ofgreater than or equal to 8.0, such as from about 8.0 to about 12.0; amethod for measuring this characteristic is set out in Example 16;

the CTLA4-Ig molecules have N-linked glycosylation such that Domain Iexhibits an area percentage of about 24.5% to about 35.2%, or Domain IIexhibits an area percentage of about 26.3% to about 34.1%, or Domain IIIexhibits an area percentage of about 21.9% to about 31.5%, or Domain IVand Domain V exhibits an area percentage of about 7.9% to about 18.6%; amethod for measuring this characteristic is set out in Example 44;

the CTLA4-Ig molecules have an average molar ratio of NGNA to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, ofless than or equal to 1.5; a method for measuring this characteristic isset out in Example 16.

The invention provides for such compositions as isolated orsubstantially purified. The invention provides for such compositions aspharmaceutical compositions or pharmaceutically acceptable compositions.The invention provides for compositions having any permutation orcombination of these characteristics.

The invention provides for such compositions as isolated orsubstantially purified. The invention provides for such compositions aspharmaceutical compositions or pharmaceutically acceptable compositions.The invention provides for compositions having any permutation orcombination of these characteristics:

(2) the CTLA4-Ig molecules comprise any one or more of SEQ ID NO: 4, 11,12, 13, 14, 15, 16 or 24 (for example CTLA4^(A29YL104E)-Ig), theCTLA4-Ig molecules are less than or equal to 5.0 area percent CTLA4-Ighigh molecular weight species (or CTLA4-Ig tetramers) as determined bysize exclusion chromatography and spectrophotometric detection (a methodof measurement for which is set out in Example 25). More particularly,the invention provides such compositions further having one or more ofthe following characteristics:

the CTLA4-Ig molecules are greater than or equal to 95.0 area percentCTLA4-Ig dimers as determined by size exclusion chromatography andspectrophotometric detection; a method for measuring this characteristicis set out in Example 25;

the CTLA4-Ig molecules are less than or equal to 1.0 area percentCTLA4-Ig low molecular weight species (or CTLA4-Ig monomers) asdetermined by size exclusion chromatography and spectrophotometricdetection; a method for measuring this characteristic is set out inExample 25;

providing (as CTLA4-Ig molecules or as said composition) about 8-15bands with a pI range of about 4.5 to about 5.6, and cumulative bandsintensity of 95%-105% at a pI range of 4.5 to 5.6; a method formeasuring this characteristic is set out in Example 22;

not exceeding a maximum amount of DNA of about 2.5 pg/mg CTLA4-Igmolecules; a method for measuring this characteristic is set out inExample 55;

not exceeding a maximum amount of Protein A of 5 ng/mg CTLA4-Igmolecules; a method for measuring this characteristic is set out inExample 53;

not exceeding a maximum amount of MCP-1 of 5 ng/mg CTLA4-Ig molecules; amethod for measuring this characteristic is set out in Example 54;

not exceeding a maximum amount of host cell protein (also known ascellular protein) of 50 ng/mg CTLA4-Ig molecules; a method for measuringthis characteristic is set out in Example 52;

not exceeding a maximum amount of bacterial endotoxin of 0.42 EU/mgCTLA4-Ig molecules; a method for measuring this characteristic is setout in Example 48;

not exceeding a maximum bioburden of 1 CFU/ml; a method for measuringthis characteristic is set out in Example 49;

not exceeding a maximum amount of Triton X-100 of 2 ppm; a method formeasuring this characteristic is set out in Example 57;

the CTLA4-Ig molecules have an average molar ratio of sialic acid toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of greater than or equal to 5.0, such as from about 5.0 toabout 9.0 or from about 5.0 to about 10.0; a method for measuring thischaracteristic is set out in Example 39;

the CTLA4-Ig molecules have an average molar ratio of NANA to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, ofgreater than or equal to 5.0, such as from about 5.0 to about 9.0 orfrom about 5.0 to about 10.0; a method for measuring this characteristicis set out in Example 39;

the CTLA4-Ig molecules have an average molar ratio of GalNAc to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 0.8 to about 4.0; a method for measuring this characteristicis set out in Example 36;

the CTLA4-Ig molecules have an average molar ratio of GlcNAc to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 14 to about 35; a method for measuring this characteristic isset out in Example 36;

the CTLA4-Ig molecules have an average molar ratio of galactose toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of from about 8.0 to about 14; a method for measuring thischaracteristic is set out in Example 35;

the CTLA4-Ig molecules have an average molar ratio of fucose to CTLA4-Igmolecules (or to CTLA4-Ig dimers), expressed as moles/mole protein, offrom about 1.7 to about 9.3; a method for measuring this characteristicis set out in Example 35;

the CTLA4-Ig molecules have an average molar ratio of mannose toCTLA4-Ig molecules (or to CTLA4-Ig dimers), expressed as moles/moleprotein, of from about 9 to about 18; a method for measuring thischaracteristic is set out in Example 35;

The invention provides for such compositions to be isolated orsubstantially purified. The invention provides for proteins andcompositions having any permutation or combination of thesecharacteristics.

Examples 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 21,28, 29, 30, 31, 32, 33, 34, 42, 44, 45, 46, 47, 48, 49, 50, 51, 58, 59,60, 61, 62, 63, 64, 65, 66, 67 above pertain in particular to theCTLA4-Ig protein of (1) above, although, as described in thisspecification, the methods relating to, and uses of, such protein inthese Examples are illustrative for methods relating to, and uses of,other CTLA4-Ig proteins of the invention.

Examples 19, 20, 22, 23, 24, 25, 26, 27, 35, 36, 37, 38, 39, 40, 41, 52,53, 54, 55, 56, 57 above pertain in particular to the CTLA4-Ig proteinof (2) above, although, as described in this specification, the methodsrelating to, and uses of, such protein in these Examples areillustrative for methods relating to, and uses of, other CTLA4-Igproteins of the invention.

1-296. (canceled)
 297. A composition comprising CTLA4^(A29YL104E)-Igmolecules comprising: (a) an average molar ratio of sialic acid toCTLA4^(A29YL104E)-Ig molecules of at least about 5; (b) an average molarratio of N-acetyl neuraminic acid (NANA) to CTLA4^(A29YL104E)Igmolecules of from about 5 to about 10; (c) an average molar ratio ofN-glycolyl neuramininic acid (NGNA) to CTLA4^(A29YL104E)-Ig moleculesless than or equal to 1.5; (d) an average molar ratio ofN-Acetylgalactosamine (GalNAc) to CTLA4^(A29YL104E)-Ig molecules fromabout 2.7 to about 3.6; (e) an average molar ratio ofN-Acetylglucosamine (GlcNAc) to CTLA4^(A29YL104E)-Ig molecules fromabout 24 to about 28; (f) an average molar ratio of galactose toCTLA4^(A29YL104E)-Ig molecules from about 9.2 to about 17; (g) anaverage molar ratio of fucose to CTLA4^(A29YL104E)-Ig molecules fromabout 4.2 to about 7.0; (h) an average molar ratio of mannose toCTLA4^(A29YL104E)-Ig molecules from about 10 to about 20; and (i) lessthan or equal to 5.0 area percent high molecular weight species asdetermined by size exclusion chromatography and spectrophotometricdetection, wherein the CTLA4^(A29YL104E)-Ig molecules comprise one ormore CTLA4^(A29YL104E)-Ig polypeptides having the amino acid sequenceset forth in SEQ ID NO: 4, 11, 12, 13, 14, 15, or
 16. 298. TheCTLA4^(A29YL104E)-Ig composition of claim 297, wherein the concentrationof MCP-1 in the CTLA4^(A29YL104E)-Ig composition is less than about 5ppm.
 299. The CTLA4^(A29YL104E)Ig composition of claim 297, wherein theCTLA4^(A29YL104E)-Ig composition comprises at least 95% of dimericCTLA4^(A29YL104E)-Ig forms.
 300. The CTLA4^(A29YL104E)-Ig composition ofclaim 297, wherein the CTLA4^(A29YL104E)-Ig composition comprises lessthan or equal to 4.0 area percent high molecular weight species asdetermined by size exclusion chromatography and spectrophotometricdetection.
 301. The CTLA4^(A29YL104E)-Ig composition of claim 297,wherein the CTLA4^(A29YL104E)-Ig composition comprises less than orequal to 3.0 area percent high molecular weight species as determined bysize exclusion chromatography and spectrophotometric detection.
 302. TheCTLA4^(A29YL104E)-Ig composition of claim 297, wherein theCTLA4^(A29YL104E)-Ig composition comprises less than or equal to 2.5area percent high molecular weight species as determined by sizeexclusion chromatography and spectrophotometric detection.
 303. TheCTLA4^(A29YL104E)-Ig composition of claim 297, wherein theCTLA4^(A29YL104E)-Ig composition comprises less than or equal to 2.0area percent high molecular weight species as determined by sizeexclusion chromatography and spectrophotometric detection.
 304. TheCTLA4^(A29YL104E)-Ig composition of claim 297, wherein theCTLA4^(A29YL104E)-Ig composition comprises a polypeptide having theamino acid sequence set forth in SEQ ID NO:
 16. 305. TheCTLA4^(A29YL104E)-Ig composition of claim 297, wherein (a) about 90% ofthe CTLA4^(A29YL104E)-Ig polypeptides are polypeptides of SEQ ID NO: 13and/or 16; (b) about 10% of the CTLA4^(A29YL104E)-Ig polypeptides arepolypeptides of SEQ ID NO: 12 and/or 15; (c) about 4% of theCTLA4^(A29YL104E)-Ig polypeptides are polypeptides of SEQ ID NO:4, 25,25, 27, or any combination thereof; and, (d) about 96% of theCTLA4^(A29YL104E)-Ig polypeptides are polypeptides of SEQ ID NO: 14, 15,16, or any combination thereof.